DEVELOPMENT OF A FOUR - STEP SEMI - BIOSYNTHESIS OF THE ANTICANCER DRUG PACLITAXEL AND ITS ANALOGUES By Chelsea Thornburg A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Bi 2015 ABSTRACT DEVELOPMENT OF A FOUR - STEP SEMI - BIOSYNTHESIS OF THE ANTICANCER DRUG PACLITAXEL AND ITS ANALOGUES By Chelsea Thornburg Paclitaxel (Taxol®) is a widely used chemotherapeutic drug wit h additional medical applications in drug - eluting stents as an anti - restenosis treatment. Paclitaxel is a structurally complex natural product with an excellent scaffold for designing analog s with pharmacological properties. To date, clinically approved an alog s include docetaxel and cabazitaxel for the treatment of additional cancers. Currently, plant cell fermentation methods produce paclitaxel and large quantities of the precursors 10 - deacetylbaccatin III (10 - DAB) and baccatin III. The complexity of the s emi - characterized ~19 - step paclitaxel biosynthetic pathway limits bioengineering attempts. However, the availability of 10 - DAB and baccatin III suggests a semi - bios ynthetic pathway to paclitaxel starting with these precursors is feasible. We have designed a short, simple biosynthetic pathway, capable of making paclitaxel, analog s, and/or valuable precursors for the semi - synthesis of additional analog s of biological interest. The paclitaxel biosynthesis enzyme baccatin III: 3 - amino - 13 - O - phenylpropanoyl CoA transferase (BAPT) and the bacterial (2 R ,3 S ) - phenylisoserinyl CoA ligase (PheAT) produce N - debenzoylpaclitaxel, N - debenzoyldocetaxel, or precursor analog s. The addition of the paclitaxel biosynthetic N - debenzoyltaxol - N - benzoyltransferase (NDTNBT) and the b acterial benzoate CoA ligase (BadA) produce paclitaxel or other N - acylated analog s. In this dissertation, BAPT and BadA are kinetically characterized. The substrate specificity of BadA was systematically investigated with a series of 24 substrates. Six cry stal structures of BadA in complex with different substrates, including benzoyl AMP, are used to explain BadA reactivity and propose rational mutations (A227A, H333A, and I334A) that expand su bstrate specificity and provide insight into the BadA mechanism and connect with established acetylation regulatory mechanisms in bacteria . Major hurdles including solubility and substrate availability, were overcome in order to characterize BAPT activity in the proposed semi - biosynthetic pathway. BAPT was purified as a fusion protein with maltose binding protein and its (2 R ,3 S ) - phenylisoserinyl CoA substrate was biosynthesized. To our knowledge this is the first time (2 R ,3 S ) - phenylisoserinyl CoA has been isolated in quantitative yields high enough to allow for charact erization of the Michaelis - Menten kinetic constant s ( k cat and K M ) for BAPT. This dissertation also describes the combination of BAPT and a bacterial ligase (PheAT) to produce N - debenzoylpaclitaxel and N - debenzoyl - 10 - deacetylpaclitaxel, precursors of paclit axel and docetaxel, respectively. Biosynthesis of a biologically active paclitaxel analog , N - 2 - furanyl - N - debenzoylpaclitaxel, using the aforementioned enzymes, is also demonstrated as proof - of - principle that this semi - biosynthetic pathway may shorten the n umber of steps required to make certain paclitaxel (and docetaxel) analogs of interest. Copyright by CHELSEA THORNBURG 2015 v ACKNOWLEDGEMENTS I would like to acknowledge my advisor, Dr. Kevin Walker, for his support during my t ime here at Michigan State University . I also would like to acknowledge Dr. Dan Jones for all his advice and assistance in learning mass spectrometry. Dr. Jim Geiger kindly trusted me with his chromatography equipment and was a great collaborator for the B adA ligase crystallography work . The biochemistry and molecular biology (BMB) department has been a wonderful academic home. Faculty members were always willing to discuss any research problems I encountered along the way. The following professors sat do wn with me at some point and I have to thank my family for all their love and support. My mom - Kristen Thornburg, my P api - Mark Santas, my sisters - Caitlin Thornburg and Rhoda Brew - Appiah, my bro - in - law Matt Seidel, and my niece M adison are always there for me even though they have no idea what I do all day. My GREAT aunt Frankie and uncle Jim welcomed me into their home and are two of my favorite people. I am also grateful t o the lovely Janelle and James Sabo (and the girls: Claire, Katherine, and Sophia) for welcoming me into their home for Thanksgiving these past few years . I also need to thank all the people I have lo st during my doctoral program. My grandfather - Newton Thornburg, my grandmother - Cloteel Atkins, my dear friend Pam Movalson and her daugh ter, Christine. I miss you all. vi TABLE OF CONTENTS LIST O F TABLES ................................ ................................ ................................ ............... x LIST OF FIGURES ................................ ................................ ................................ ........... xi KEY TO ABBREVIATIONS ................................ ................................ ......................... xvii Chapt er 1. Clinical use and production of paclitaxel and analogs of clinical interest ......... 1 1.1 Introduction ................................ ................................ ................................ ........... 1 1.1.1 Clinical uses of pac litaxel ................................ ................................ ............. 1 1.1.2 Clinical uses of paclitaxel analogs ................................ ................................ 3 1.1.3 A brief history of paclitaxel ................................ ................................ .......... 3 1.1.4 Paclitaxel mode of action ................................ ................................ .............. 5 1.1.5 Paclitaxel biosynthesis ................................ ................................ .................. 7 1.1.6 Paclitaxel production ................................ ................................ .................. 11 1.1.7 Semi - biosynthesis of paclitaxel, precursors, and analogs ........................... 14 REFERENCES ................................ ................................ ................................ ............... 19 Chapter 2. Kinet ically - and crystallographically - guided mutations of a benzoate CoA ligase (BadA) elucidate mechanism and expand substrate permissivity ........................... 33 2.1 Introduction ................................ ................................ ................................ ......... 33 2.2 Experimental ................................ ................................ ................................ ....... 38 2.2.1 Materials ................................ ................................ ................................ ..... 38 2.2.2 Plasmids ................................ ................................ ................................ ...... 39 2.2.3 BadA protein expression and purification ................................ .................. 39 2.2.4 BadA kinetic assays ................................ ................................ .................... 40 2.2.5 BadA assay analysis by liquid chromatography mass spectrometry .......... 41 2.2.6 BadA mutations ................................ ................................ .......................... 42 2.2.7 Kinetic analysis ................................ ................................ ........................... 43 2.2.8 BadA crystal structures ................................ ................................ ............... 43 2.2.8.1 Crystallization of R. palustris benzoate: coenzyme A ligase (BadA) ...... 43 2.2.8.2 Co - crystallization to obtain the ligand bound structure ........................... 44 2.2.8.3 Data processing and refinement of BadA ................................ ................ 44 2.2.9 Calculation of covalent van der Waals volumes and lengths ..................... 45 2.3 Results ................................ ................................ ................................ ................. 46 2.3.1 Solving the BadA structure ................................ ................................ ......... 46 2.3.1.1 Domain orientation ................................ ................................ ................... 46 2.3.1.2 Features of the BadA active site ................................ ............................... 47 2.3.2 Kinetic properties of BadA ................................ ................................ ......... 50 2.3.3 Substrate turnover by BadA ................................ ................................ ........ 50 2.3.3.1 Halogenated benzoates ................................ ................................ ............. 50 2.3.3.2 Benzoates with strongly electron - withdrawing substituents .................... 52 2.3.3.3 Benzoates with strongly electron - donating substituents .......................... 52 vii 2.3.3.4 Turnover of heteroaromatic carboxylates ................................ ................ 53 2.3.3.5 Turnover of non - aromatic carbocycle carboxylates ................................ . 54 2.3.4 Rational Mutation of the BadA Active Site ................................ ................ 54 2.3.4.1 Ala227Gly - BadA mutant ................................ ................................ ......... 56 2.3.4.2 Ile334Ala - BadA mutant ................................ ................................ ........... 56 2.3.4.3 His333Ala - BadA mutant ................................ ................................ .......... 57 2.3.4.4 Leu332Ala - BadA mutant ................................ ................................ ......... 57 2.4 Discussion ................................ ................................ ................................ ........... 58 2.4.1 BadA structure and homology ................................ ................................ .... 58 2.4.2 Catalytically important lysine residues in BadA ................................ ........ 62 2.4.3 Structural rationale for substrate specificity of BadA ................................ . 64 2.4.3.1 Non - aromatic carbocycle carboxylates ................................ .................... 67 2.4.4 Analysis of point mutants of BadA ................................ ............................. 68 2.5 Conclusions ................................ ................................ ................................ ......... 70 APPENDI X ................................ ................................ ................................ .................... 72 REFERENCES ................................ ................................ ................................ ............... 93 Chapter 3. Expression and purification of the Taxus cuspidata baccatin III: 3 - amino - 13 - O - phenylpropanoyl tra nsferase (BAPT) ................................ ................................ ................ 99 3.1 Introduction ................................ ................................ ................................ ......... 99 3.2 Experimental ................................ ................................ ................................ ..... 107 3.2. 1 Materials ................................ ................................ ................................ ... 107 3.2.2 BAPT purification from Pichia pastoris ................................ ................... 107 3.2.2.1 Cloning of pHisBAPT and pMycBAPT ................................ ................ 107 3.2.2.2 BAPT expression in P. pastoris ................................ ............................. 109 3.2.2.3 BAPT Western blot ................................ ................................ ................ 109 3.2.3 BAP T purification from Escherichia coli ................................ ................. 110 3.2.3.1 Cloning of pNterBAPT ................................ ................................ .......... 110 3.2.3.2 Cloning of pCterBAPT ................................ ................................ ........... 110 3.2.3.3 Cloning of pOptBapt ................................ ................................ .............. 111 3.2.3.4 Cloning of pMBP - CterBAPT and pMBP - NterBAPT ............................ 111 3.2.3.5 E. coli strains ................................ ................................ .......................... 112 3.2.3.6 BAPT expression in E. coli ................................ ................................ .... 112 3.2.3.7 Nickel - affinity chromatography ................................ ............................. 113 3.2.3.8 Ammonium sulfate fractionation ................................ ........................... 114 3.2.3.9 Ion exchange chromatography ................................ ............................... 114 3.2.4 Optimized NterBAPT expression and purification. ................................ .. 115 3.2.5 MBP - BAPT expression and purification ................................ .................. 115 3.2.6 Band densitome try ................................ ................................ .................... 116 3.2.7 Proteomics. ................................ ................................ ................................ 117 3.2.8 BAPT activity assays ................................ ................................ ................ 1 17 3.3 Results and discussion ................................ ................................ ....................... 119 3.3.1 BAPT expression in Pichia pastoris ................................ ......................... 119 3.3.2 BAPT expression and purification in E. coli ................................ ............ 122 3.3.2.1 Expression optimization in E. coli ................................ ......................... 122 3.3.2.2 Purification by nickel - affinity chromatography ................................ ..... 125 viii 3.3.2.3 Ammonium sulfate precipitation ................................ ............................ 127 3.3.2.4 Ion exchange chromatography ................................ ............................... 127 3.3. 3 Optimized NterBAPT purification ................................ ............................ 128 3.3.4 BAPT activity assays ................................ ................................ ................ 131 3.3.5 MBP - BAPT expression and purification ................................ .................. 135 3.4 Conclusions ................................ ................................ ................................ ....... 138 3.5 Future Research ................................ ................................ ................................ . 140 APPENDIX ................................ ................................ ................................ .................. 141 REFERENCES ................................ ................................ ................................ ............. 147 Chapter 4. Paclitaxel analog biosynthesis from baccatin III with a four - enzyme in vitro system and characterization of baccatin III - 3 - amino - 13 - O - phenylpropanoyl CoA transferase (BAPT) ................................ ................................ ................................ .......... 153 4.1 Introduction ................................ ................................ ................................ ....... 153 4.1.1 Engineering the paclitaxel biosynthetic path way ................................ ..... 153 4.1.2 Proposed paclitaxel semi - biosynthesis ................................ ..................... 160 4.1. 3 Applications in paclitaxel analog production ................................ ........... 162 4.1. 4 BAPT characterization and proof - of - principle for paclitaxel/analog biosynthesis ................................ ................................ ................................ ............. 163 4.2 Experimental ................................ ................................ ................................ ..... 164 4.2.1 Materials ................................ ................................ ................................ ... 164 4.2.2 Chemical synthesis of (3 R ) - - phenylalanyl CoA ................................ ..... 164 4.2.3 Method I. Synthe sis of (2 R ,3 S ) - phenylisoserinyl CoA ............................. 166 4.2.4 Method II. Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA ........................... 167 4.2.5 Method III. Synthes is of (2 R ,3 S ) - phenylisoserinyl CoA .......................... 167 4.2.6 Method IV. Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA .......................... 168 4.2.7 PheAT purification ................................ ................................ .................... 169 4.2.8 Biosynthesis of (2 R ,3 S ) - phenylisoserinyl CoA ................................ ........ 169 4.2.9 Purification of (2 R ,3 S ) - phenylisoserinyl CoA ................................ .......... 170 4.2.10 HPLC Analysis of acyl CoA thioesters ................................ .................... 171 4.2.11 Acyl CoA purity analysis (Ellman assay) ................................ ................. 172 4.2.12 BAPT, BadA, and NDTNBT purification ................................ ................ 173 4.2.13 BAPT kinetic assays ................................ ................................ ................. 173 4.2.14 Kinetic analysis ................................ ................................ ......................... 174 4.2.15 Liquid chromatography mass spectrometry: BAPT assay analysis .......... 174 4.2.16 PheAT and BAPT coupled reactions ................................ ........................ 175 4.2.17 Production of paclitaxel and its analogs in a coupled enzyme assay ........ 175 4.3 Results and discussion ................................ ................................ ....................... 177 4.3.1 Synthesis of acyl CoA substrates ................................ .............................. 177 4.3.1.1 Synthesis of (3 R ) - - phenylalanyl CoA ................................ .................. 178 4.3.1.2 Synthesis of (2 R ,3 S ) - Phenylisoserinyl CoA ................................ .......... 179 4.3.2 Biosynthesis of (2 R ,3 S ) - PhIS CoA. ................................ .......................... 182 4.3.3 Michaelis - Menten kinetics of BAPT ................................ ........................ 184 4.3.3.1 (3 R ) - - phenylalanyl CoA and baccatin III substrates ............................ 184 4.3.3.2 (2 R ,3 S ) - Phenylisoserinyl CoA and baccatin III substrates .................... 186 ix 4.3.3.3 (2 R ,3 S ) - Phenylisoserinyl CoA and 10 - deacetylbaccatin III as substrates ................................ ................................ ................................ ............... 186 4.3.4 BAPT activity and substrate assisted catalysis ................................ ......... 187 4.3.5 Coupled biosynthesis of N - debenzoylpaclitaxel: PheAT and BAPT. ...... 193 4.3.6 Biosynthesis of N - 2 - furanoyl - N - de benzoylpaclitaxel: a paclitaxel analog ................................ ................................ ................................ .... 194 4.4 Conclusions ................................ ................................ ................................ ....... 197 4.5 Future Research ................................ ................................ ................................ . 199 APPENDIX ................................ ................................ ................................ .................. 201 REFERENCES ................................ ................................ ................................ ............. 223 x LIST OF TABLES Table 1.1. Clinically - approved taxanes, their current uses, and open clinical trials. .......... 3 Table 2.1. Kinetic p arameters of BadA for various substrates. ................................ ........ 51 Table 2.2. Relative apparent maximum rates of BadA and point mutants for various substrates. ................................ ................................ ................................ .......................... 55 Table 3.1. Biologically active analogs as active as paclitaxel with modifications in the C13 - phenylisoserinyl sidechain. ................................ ................................ ............................. 106 Table 3.2. Proteomics analysis of purified NterBAPT. ................................ .................. 130 Table 3.3. Proteomics analysis of purified MBP - BAPT. ................................ ................ 137 Table 4.1. Simple analogs of paclitaxel with biological activity. ................................ ... 162 Table 4.2. Kinetic constants of enzymes required for proposed paclitaxel biosynthesis from baccatin III. ................................ ................................ ................................ ..................... 187 Table 4.3. Michae lis - Menten kinetic constants from BAHD acyltransferase family members. ................................ ................................ ................................ ......................... 191 xi LIST OF FIGURES Figure 1.1. Clinically - approved taxanes ................................ ................................ .............. 2 Figure 1.2. Paclitaxel mode of action ................................ ................................ .................. 5 Figure 1.3. Taxoid deriva tives of taxa - 4(5),11(12) - diene ................................ .................... 7 Figure 1.4. Paclitaxel biosynthesis: Part 1 ................................ ................................ ........... 8 Figure 1.5. Paclitaxel biosynthesis: Part 2 ................................ ................................ ......... 10 Figure 1.6. Examples of off - pathway metabolites that divert flux away from paclitaxel production ................................ ................................ ................................ .......................... 11 Figure 1.7. Semi - synt hesis of paclitaxel from 10 - DAB ................................ .................... 13 Figure 1.8. Taxanes clinically approved and in clinical trials ................................ ........... 14 Figure 1.9. Coupled enzyme biosynthesis of paclitaxel. ................................ ................... 15 Figu re 2.1. Representative natural products derived from ac yl CoA thioesters ................ 34 Figure 2.2. ATP - dependent, two - step mechanism of a benzoate: CoA ligase in the presence of magnesium ................................ ................................ ................................ ...... 36 Figure 2.3. Acyl CoA detection by tandem mass spectrometry ................................ ........ 41 Figure 2.4. Crystal structure of BadA in complex with benzoate ................................ ...... 46 Figure 2.5. The active site of the BadA structure ................................ .............................. 49 Figure 2.6. BadA structural alignments an d C - terminal domain orientation ..................... 61 Figure 2.7. Active site of BadA showing possible polar contacts between Lys427 and Bz - AMP. ................................ ................................ ................................ ................................ .. 63 Figure A.1.1. Michaelis - Menten plot of biosyn thetic benzoyl CoA catalyzed by BadA. . 73 Figure A.1.2. Michaelis - Menten plot of biosynthetic thiophene - 2 - carbonyl CoA catalyzed by BadA ................................ ................................ ................................ ............................. 73 Figure A.1.3. Michaelis - Menten plot of biosynthetic 3 - furoyl CoA catalyzed by BadA .. 74 Figure A.1.4. Michaelis - Menten plot of biosynthetic cyclohexanoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 74 xii Figure A.1.5. Michaelis - Menten plot of biosynthetic 1 - cyclohexen - 1 - oyl CoA catalyzed by BadA. ................................ ................................ ................................ ................................ . 75 Figure A.1.6. Michaelis - Menten plot of biosynthetic 3 - cyclohexen - 1 - oyl CoA catalyzed by BadA. ................................ ................................ ................................ ................................ . 75 Figure A.1.7. Michaelis - Menten plot of biosynthetic 2 - fluorobenzoyl CoA catalyzed by BadA. ................................ ................................ ................................ ................................ . 76 Figure A.1.8. Michaelis - Menten plot of biosynthetic 3 - fluorobenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 76 Figure A.1.9. Michaelis - Menten plot of biosynthetic 4 - fluorobenzoyl CoA catalyzed by BadA. ................................ ................................ ................................ ................................ . 77 Figure A.1.10. Michaelis - Menten plot of biosynthetic 2 - chlorobenzoyl CoA catalyzed by BadA. ................................ ................................ ................................ ................................ . 77 Figure A.1 .11. Michaelis - Menten plot of biosynthetic 3 - chlorobenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 78 Figure A.1.12. Michaelis - Menten plot of biosynthetic 4 - chlorobenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 78 Figure A.1.13. Michaelis - Menten plot of biosynthetic 2 - aminobenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 79 Figure A.1.14. Michaelis - Menten plot of biosynthe tic 3 - aminobenzoate CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 79 Figure A.1.15. Michaelis - Menten plot of biosynthetic 4 - aminobenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 80 Figure A.1.16. Michaelis - Menten plot of biosynthetic 2 - hydroxybenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 80 Figure A.1.17. Michaelis - Menten plot of biosynthetic 3 - hydroxybenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 81 Figure A.1.18. Michaelis - Menten plot of biosynthetic 4 - hydroxybenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 81 Figure A.1.19. Michaelis - Menten plot of biosynthetic 2 - methylbenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 82 Figure A.1.20. Michaelis - Menten plot of biosynthetic 3 - methylbenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 82 xiii Figure A.1.21. Michaelis - Menten plot of biosynthetic 4 - methylbenzoate CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 8 3 Figure A.1.22. Michaelis - Menten plot of biosynthetic 2 - cyanobe nzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 83 Figure A.1.23. Michaelis - Menten plot of biosynthetic 2 - methoxybenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ............................. 84 Fi gure A.1.24. Michaelis - Menten plot of biosynthetic 2 - nitrobenzoyl CoA catalyzed by BadA ................................ ................................ ................................ ................................ .. 84 Figure A.1.25. Crystals of Rhodopseudomonas palustris benzoate CoA ligase ............... 85 Figure A.1.26. Overlay of BadA structures in complex with bound ligands. .................... 85 Figure A.1.27. Michaelis - Menten plot of benzoyl CoA catalyzed by the Lys427A la - BadA mutant. ................................ ................................ ................................ ............................... 86 Figure A.1.28. Benzoyl AMP (Bz - AMP) produced by BadA and the Lys427Ala - BadA mutant ................................ ................................ ................................ ................................ 87 Figure A.1.29. Shifted position of Tyr432 in BadA bound with bzAMP .......................... 88 Figure A.1.30. Partial amino acid sequence alignment of selected benzoate CoA ligases. ................................ ................................ ................................ ................................ 89 Figure A.1.31. Comparison of the position of Phe226 of BadA (in the thiolation conformation) and that of Phe236 of BCL M (in the adenylation conformation) bound to benzoate ................................ ................................ ................................ ............................. 89 Figure A.1.32. Partial amino acid sequence alignment of a broad selection of ATP - dependent adenylases and coenzyme A ligases. ................................ ................................ 90 Figure A.1.33. Relative position of benzoate and benzoyl AMP in the Ba dA active site. 90 Figure 3.1. Proposed mechanism of BAHD acyltransferases ................................ .......... 100 Figure 3.2. Examples of BAHD enzyme products. ................................ ......................... 101 Figure 3.3. BAHD structure of the Sorghum hydroxycinnamoyltransferase (HCT) ....... 102 Figure 3.4. Condensed paclitaxel biosynthesis f rom geranylgeranyl diphosphate showing the BAHD acyltransferases in the pathway. ................................ ................................ .... 103 Figure 3.5. SDS - PAGE of whole cell lysate from expressions of BAPT clones from P. pastoris ................................ ................................ ................................ ............................. 120 Figure 3.6. NterBAPT expression in crude cell lysates. ................................ .................. 121 xiv Figure 3.7. Soluble expression of NterBAPT with varied IPTG concentrations ............. 122 Figure 3.8. Expression and induction of OptBAPT in E. coli ................................ ......... 123 Figure 3.9. Relative expression levels of NterBAPT and the Taxus N - benzoylt ransferase (NDTNBT) by SDS - PAGE ................................ ................................ .............................. 124 Figure 3.10. SDS - PAGE of NterBAPT purification by nickel - affinity chromatography 125 Figure 3.11. NterBAPT activity during nick el - affinity chromatography ........................ 126 Figure 3.12. Ammonium sulfate fractionation of NterBAPT clarified lysate ................. 127 Figure 3.13. Purification of NterBAPT b y multiple chromatography steps .................... 129 Figure 3.14. Coupled assa y schematic with PheAT and BAPT ................................ ...... 131 Figure 3.15. Biosynthesis of N - deben zoyl - - deoxypaclitaxel by BAPT ........................ 132 Figure 3.16. Representati ve LC - ESI/MS/MS of a BAPT assay . ................................ ..... 134 Figure 3.17. Domain structures of MBP - BAPT fusion proteins ................................ ..... 135 Figure 3.18. Representative p urification of MBP - BAPT . ................................ ............... 136 Figure A.2.1. Multiple sequence alignment of Taxus cuspidata acyltransferases ........... 142 Figure A.2.2. HisBAPT amino acid sequence for expression in P. pastoris ................... 143 Figure A.2.3. cMycHisBAPT amino acid sequence for expression in P. pastoris . ........ 143 Figure A.2.4. NterBAPT amino acid sequence for exp ression in E. coli . ....................... 143 Figure A.2.5. CterBAPT amino acid sequence for expression in E. coli ........................ 143 Figure A.2.6. Codon optimized b apt gene sequence ................................ ....................... 144 Figure A.2.7. MBP - CterBAPT (95.2 kDa) amino acid sequence for expression in E. coli ................................ ................................ ................................ ................................ .... 145 Figure A.2.8. Quan tification of BAPT by band densitometry. ................................ ........ 145 Figure A.2.9. SDS - PAGE gel of purified NterBAPT and MBP - CterBAPT for proteomics analysis. ................................ ................................ ................................ ............................ 146 Figure 4.1. Condensed sc heme of paclitaxel biosynthesis ................................ ............... 155 Figure 4.2. Products of an engineered chimeric cytochrome P450 taxadiene - - hydroxylase expressed in E. coli ................................ ................................ ...................... 157 xv Figure 4.3. Coupled enzyme biosynth esis of paclitaxel ................................ .................. 159 Figure 4.4. Taxanes clinically approved and in clinical trials ................................ ......... 161 Figure 4.5. Structures of (3 R ) - - phenylalanyl coenzyme A (R = H) and (2 R ,3 S ) - phenylisoserinyl coenzyme A (R = OH). ................................ ................................ ......... 177 Figure 4.6. Organic synthesis of (3 R ) - - Phenylalanyl CoA ................................ ............ 1 79 Figure 4.7 . Conformation of (2 R ,3 S ) - phenylisoserine ................................ ..................... 181 Figure 4.8 . Biosynthesis of (2 R ,3 S ) - phenylisoserinyl CoA with PheAT, a truncated form of tyrocidine synthetase A (TycA) ................................ ................................ ................... 182 Figure 4.9 . Biosynthetic reaction progress curve for (2 R ,3 S ) - PhIS CoA ........................ 183 Figure 4.10 . BAPT - catalyzed enzyme reaction ................................ .............................. 184 Figure 4.11 . Multiple sequence alignment of bapt genes from Taxus sp. ....................... 188 Figure 4.12 . Proposed mechanisms for BAHD acyltransferases and BAPT ................... 189 Figure 4.13 . Homology model of BAPT based on the hydroxycinnamoyl transferase (PDB: 4G0B) from Coffea canephora ................................ ................................ ............. 192 Figure 4.14 . N - deb enzoylpaclitaxel biosynthesized from BAPT and PheAT coupled reactions ................................ ................................ ................................ ........................... 193 Figure 4.15 . Biosynthesis of N - 2 - furanoyl - N - debenzoylpaclitaxel with PheAT, BAPT, BadA, and NDTNBT ................................ ................................ ................................ ....... 194 Figure 4.16 . Overlapping substrate specificities o f PheAT, BAPT, BadA, and NDTNBT ................................ ................................ ................................ ......................... 196 Figure A.3.1. 1 H - NMR of R - N - Boc - 3 - amino - 3 - phenylpropanoic acid. .......................... 202 Figure A.3.2. 1 H - NMR of chemically synthesized (3 R ) - - phenylalanyl CoA. ............... 203 Figure A.3.3. HPLC chromatogram of purifi ed (3 R ) - - phenylalanyl CoA. .................... 204 Figure A.3.4. Mass spectra of purified (3 R ) - - phenylalanyl CoA ................................ .. 205 Figure A.3.5. LC - ESI/MSMS chr omatogram showing the production of N - debenzoyl - - deoxypaclitaxel. ................................ ................................ ................................ ............... 206 Figure A.3.6. (Scheme I) Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA . .......................... 206 Figure A.3.7. (Scheme II) (2 R ,3 S ) - PhIS CoA synthesis with 2 - OH protection . ............. 207 xvi Figure A.3.8. (Scheme III) (2 R ,3 S ) - PhIS CoA synthesis by DCC/ HOBt coupling . ...... 2 07 Figure A.3.9 . (Scheme IV) (2 R ,3 S ) - PhIS CoA synthesis by DCC/NHS coupling . ......... 20 8 Figure A.3.10 . 1 H - NMR of ( 2 R, 3 S) - N - Boc - 3 - amino - 3 - phenylisoserine. ........................ 209 Figure A.3.11 . 1 H - NMR of biosynthetic (2 R ,3 S ) - phenylisoserinyl CoA. ....................... 210 Figure A.3.12 . 13 C - NMR of biosynthetic (2 R ,3 S ) - phenylisoserinyl CoA. ...................... 211 Figur e A.3.13 . HPLC analysis of purified (2 R ,3 S ) - phenylisoserinyl CoA. ..................... 212 Figure A.3.14 . Mass spectra of purified (2 R ,3 S ) - phenylalanyl CoA ............................... 213 Figure A.3.15 . Michaelis - Menten kinetic plot of biosynthetic N - debenzoylpaclitaxel produced by MBP - BAPT ................................ ................................ ................................ . 214 Figure A.3.1 6 . Michaelis - Menten kinetic plot of biosynthetic N - debenzoyl - - deoxypaclitaxel produced by MBP - BAPT ................................ ................................ ...... 214 Figure A.3.17 . Michaelis - Menten kinetic plot of biosynthetic N - debenzoyl - - deoxypaclitaxel produced by MBP - BAPT. ................................ ................................ ..... 215 Figure A.3.18 . BAPT activity with 10 - DAB and (2 R ,3 S ) - PhIS CoA .............................. 216 Figure A.3.19 . Purified recombinant PheAT ................................ ................................ ... 217 Figure A.3.20 . Production of N - debenzoyl - - deoxypaclitaxel by BAPT. ..................... 218 Figure A.3.21 . Production of N - debenzoylpaclitaxel by BAPT. ................................ ..... 220 Figure A.3.22 . BAPT and PheAT coupled assay production of N - debenzoylpaclitaxel. 221 Figure A.3. 23 . Multi - enzyme biosynthesis of N - furanoyl - N - debenzoylpaclitaxel. ......... 222 xvii KEY TO ABBREVIATIONS (2 R ,3 S ) - PhIS CoA (2 R ,3 S ) - phenylisoserinyl CoA (3 R ) - - Phe CoA (3 R ) - - p henylalanyl CoA 4CL 4 - c hlorobenzoate CoA ligase ( Alcaligenes sp .) 10 - DAB 10 - deacetylbaccatin III Å a ngstrom A a bsorbance Ac a cetyl Aq a queous AIDS a cquired immune deficiency syndrome AMP a denosine monophospha te ATP a denosine triphosphate BadA b enzoate CoA ligase ( Rhodopseudomonas palustris ) BAPT baccatin III: 3 - amino - 13 - O - phenylpropanoyl CoA transferase BCL M b enzoate CoA ligase ( Burkholderia xenovorans ) Bz b enzoyl BSA b ovine serum albumin Bz O b enzoate CAN c erium(IV) ammonium nitrate xviii CH 2 Cl 2 m ethylene chloride CoA coenzym e A Cyc cyclohexane carboxylic acid 1 - Cyc 1 - cyclohexen - 1 - carboxylic acid 3 - Cyc 3 - cyclohexen - 1 - carboxylic acid DBAT 10 - deacetylbaccatin III 10 - O - acetyltr ansferase DTT dithiothreitol DMADP dimethylallyldiphosphate EDTA ethylenediaminetetraacetic acid EtOAc ethyl acetate h h our GTP guanos ine triphosphate H 2 O water HCl hyd rochloric acid HEPES 4 - (2 - hydroxyethyl) - 1 - piperazineethanes ulfonic acid HPLC h igh performance liquid chromatography IDP i sopentenyl diphosphate IPTG - D - 1 - thiogalactopyranoside kDa k ilodalton LB Luria Bertani NHS N - h ydroxysuccinimide ester OD o ptical density xix MBP m altose binding protein MeOH m ethanol min m inute MS m ass spectrometry MOPS 3 - ( N - morpholino)propanesulfoni c acid NDTNBT N - debenzoyltaxol - N - benzoyltransferase NMR nuclear magnetic resonance PCF pl ant cell fermentation PCR p olymerase chain reaction Ph p henyl PheAT p henylisoserine CoA ligase PMSF p henylmethanesulfonylfluoride PP i i norganic phosphate RPM r evolutions per minute r. t . r oom temperature s s econd SDS - PAGE s odium docecylsulfate polyacrylamide gel electrophoresis TES t riethylsilyl THF t etrahydrofuran TLC t hin layer chromatography TS taxadiene synthase UV u ltrav iolet 1 Chapter 1. C linical use and produc tion of paclitaxel and analogs of clinical interest 1.1 Introduction Cancer is a leading cause of disease and death worldwide ( 1 ) . In the United States, the ten most common forms include cancers of the breast, lung, prostate, c olon and rectum, bladder, thyroid, kidney, endometrium, and pancreas as well as melanoma and leukemia ( 1 ) . The most lethal of these cancers are certain pancreatic, lung, leukemia, and colorectal cancers with mortality rates between 30% and 80% ( 2 ) . Paclita xel and the analog s docetaxel and cabazitaxel are approved chemotherapy drugs for use with many types of cancer. Novel preparations of these compounds and other analog s show promise for treating previously chemoresistant cancers ( 3 ) . 1.1.1 Clinical uses of pac litaxel Since paclitaxel (Taxol ® ) was originally approved as a chemotherapeutic for hormone - refractory ovarian cancer in 1992, its usage has expanded to include treatment for a wide variety of cancers including metastatic breast cancer, non - small cell lung cancer, and AIDS - ( 1 ) . More recently, paclitaxel was approved as nano - particle albumin - bound (nab) - paclitaxel (under the brand name Abraxane) in combination with gemcitabine (a nucleoside analog) for the first - line treatment of pan creatic metastatic adenocarcinoma ( 3, 4 ) . Conjugated nab - paclitaxel was designed to facilitate greater drug solubility and uptake by tumor cells (33%) over sb - paclitaxel (paclitaxel solubilized in Cremophor ® EL formulation) ( 5 ) . 2 Paclitaxel is also used of f - label for the treatment of endometrial ( 6 ) , cervical ( 7 ) , and gastroesophageal cancers ( 8 - 10 ) . There are currently 345 open clinical trials for paclitaxel and nab - paclitaxel in the United States ( Table 1 . 1 ) ( 11 ) . Many of these clinical trials are for paclitaxel in combination therapies with other drugs for treating different breast, ovarian, cervical, pancreatic, melanoma, non - small cell lung, cutaneous squamous cell carcinoma, and bile duct ( cholangiocarcinoma) c ancers among others ( 11 ) . Non - cancer applications with paclitaxel include its use as a coating in drug - eluting stents to prevent in - stent restenosis in heart disease patients ( 12 - 14 ) . Usage at lower doses shows activity with other diseases ( 15 ) . In partic ular, paclitaxel shows preliminary activity preventing pulmonary fibrosis ( 16 ) , inflammation ( 17, 18 ) , and axon regeneration ( 19, 20 ) . The broad chemotherapeutic activity of paclitaxel and its growing usage in other diseases stands to increase its demand ( 21 ) . Figure 1 . 1 . Clinically - approved taxanes. Brand names are shown in parentheses. 3 1.1.2 Clinical uses of paclitaxel analogs Docetaxel is a semi - synthetic analog of paclitaxel w i th modifications at the C10 and the Figure 1 . 1 ). Docetaxel was originally approved for the treatment of non - small cell lung cancer in 1999 ( 1 ) . It was subsequently approved for the treatmen t of prostate, breast, gastric, and head and neck cancers ( 1 ) . In 2010, cabazitaxel, a second a second - line treatment for castration - resistant hormone refractory pros tate cancer ( Figure 1 . 1 ) ( 22 ) . There are currently 143 open clinical trials for docetaxel and 11 for cabazitaxel in the United States ( Table 1 . 1 ) ( 11 ) . Many of these clinic al trials are for first or second - line combination therapies in different types of cancer such as prostate, head and neck, urothelial cell, breast, solid tumor, and stomach cancers. Table 1 . 1 . Clinically - appr oved taxanes, their current uses, and open clinical trials. Drug Currently Approved Uses Clinical Trials 1 United States Worldwide Paclitaxel Ovarian, breast, non - small cell lung cancers and AIDS - 240 783 Nab - paclitaxel Breas t, non - small cell lung, and pancreatic cancer 105 152 Docetaxel Breast, prostate, non - small cell lung, stomach, and head and neck cancers 143 461 Cabazitaxel prostate cancer 11 36 1 Numbers represent clinical trials that are open and/or currently ac cepting patients. Clinical trial information was obtained from the National Institutes of Health (www.clinicaltrials.gov) 1.1.3 A brief history of paclitaxel A cytotoxic extract from the Pacific yew, Taxus brevifolia was discovered in 1964 and years later the active ingredient was identified as Taxol® (paclitaxel) ( 23, 24 ) . Early studies showed that paclitaxel blocked cell division in the G 2 and/or M phase of mitosis by promoting microtubule polymerization unlike other mitotic inhibitors (at the time) which 4 pr omoted microtubule disassembly ( 25 - 27 ) . By the late 1980s, clinical trials were underway and paclitaxel was approved for hormone - refractory ovarian cancer in 1992 and breast cancer in 1994 ( 28 ) . Successful clinical use of paclitaxel hinged on a steady dru g supply. Paclitaxel is a highly complex diterpenoid with 11 chiral centers and 5 acylations ( Figure 1 . 1 ). Because of this complexity there was no simple synthetic route available to produce commercial quantities o f the drug. Instead, paclitaxel was isolated from its natural source, the bark of the slow - growing Pacific yew tree. Isolating commercial quantities of paclitaxel from the Pacific yew proved unsustainable and fraught with environmental concerns including h abitat destruction of the threatened northern spotted owl ( 23 ) . Eventually, a total organic synthesis for paclitaxel was developed, requiring approximately 40 reaction steps ( 29 - 35 ) . Because of the large number of steps required, the total synthesis was no t cost - effective and a semi - synthetic method was sought ( 23, 36 ) . The clinical development of paclitaxel was aided by a Cooperative Research and Development Agreement (CRADA) between the National Institutes of Health (NIH) and Bristol Myers Squibb (BMS). I n return for licensing rights, BMS supported the production of paclitaxel from yew, the development of a semi - synthetic commercial production method, developed paclitaxel formulation for clinical use, and organized large - scale clinical trials ( 23 ) . The use and importance of paclitaxel are expected to increase as cancer treatments are more available worldwide ( 37 ) . 5 Figure 1 . 2 . Paclitaxel mode of action. - bound microtubule. Reprinted with permission from Macmillan Publishers Ltd: Nat. Rev. Drug Disc . 9, 790 - 803, 2010. 1.1.4 Paclitaxel mode of action Paclitaxel is active against a broad range of cancers. The accepted pharmacological mode of action suggests paclitaxel stabilizes microtubules causing cell cycle arrest and subsequent cell death ( 26, 27, 38 ) Figure 1 . 2 ) ( 39, 40 ) . As cells grow and divide, microtubules dynamically polymerize and depolymerize, facilitating normal chromosomal segregation and intracellular transport ( 40 ) . This dynamic i nstability of microtubules requires GTP - hydrolysis to function ( 39 - 41 ) . Structural studies - tubulin allosterically - and thus depolymerization ( 41 ) . Although prior studies report paclitaxel arrests cancer cell cycle leading to cell death, more recent experiments demonstrated that paclitaxel activity in 6 tumors generated multiple mitotic spindles, resulting in polar body formation, impaired cytokinesis, and ultimately apoptosis ( 42, 43 ) . While paclitaxel ultimately causes cell death in many cancer cell types, paclitaxel resistance is well - documented and can be an acquired or innate characteristic of a particular cancer ty pe ( 44 - 46 ) . Mechanisms of drug resistance include P - glycoprotein efflux pump expression ( 47 ) , altered expression of regulatory proteins ( 45, 48 ) , as well as microtubule - - tubulin isotypes, and chemi cal modification of tubulin (reviewed in ( 44 ) ). In an effort to overcome paclitaxel resistance, numerous analog s were synthesized based upon structure and function studies ( 49 - 53 ) . Modifications to the paclitaxel structure alter its reactivity and drug res istance in cancer cells. For example, the analog docetaxel ( 54 ) is more toxic than paclitaxel and is approved for use in additional cancer types such as prostate cancer. Yet another paclitaxel analog , cabazitaxel, was approved as a second - line treatment fo r hormone - refractory prostate cancer in patients already treated with docetaxel ( 22 ) . 7 Figure 1 . 3 . Taxoid derivatives of taxa - 4(5),11(12) - diene. Taxadiene synthase (TS) cyc lizes GGDP to make taxa - 4(5),11(12) - diene, which is a precursor for many different enzymes including those on the paclitaxel pathway. Several downstream biosynthetic products are shown to illustrate the diversity and complexity of the taxoids. 1.1.5 Paclitaxel biosynthesis Interest in paclitaxel biosynthesis is largely driven by efforts to optimize plant cell fermentation (PCF) technologies to produce biosynthetic paclitaxel ( 21, 55 - 58 ) . While the downstream, second half of the pathway is well - characterized, th e first half is only partially characterized ( 59, 60 ) . The pathway is highly complex with hundreds of taxoids derived from the precursor taxa - 4(5),11(12) - diene ( Figure 1 . 3 ) ( 59, 61, 62 ) . Many of the enzymes involve d in paclitaxel biosynthesis are identified, but reaction order and substrate specificities of individual enzymes remain relatively unknown, particularly in early 8 Figure 1 . 4 . Paclitaxel biosynthesis: Part 1. Enzyme abbreviations are as follows. IPPI, isopentenyl diphosphate isomerase, GGD taxadiene - 5 - - - - hydroxylase, TAT, taxadiene - - ol - O - acetyltransferase - - - - hydroxylase, - - - - - - hydroxylase, TBT, taxane - - O - benzoyltransferase, epoxidase*, oxomutase*, C9 - oxidase*, Enzymes marked with (*) are unidentified. Compound abbreviations: isopentenyl diphosphate (I DP), dimethylallyl diphosphate (DMADP), geranylgeranyl diphosphate (GGDP), 10 - deacetylbaccatin III (10 - DAB). pathway enzymes ( 63 - 65 ) . Here, a general outline of the paclitaxel biosynthetic pathway is presented. The primary metabolite precursors, isopenten yl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), are derived from the methylerythritol pathway in the plastid as opposed to the mevalonate pathway in the cytosol ( 66 ) . These isoprenoids are sequentially coupled to produce geranylgeranyl diphosph ate (GGDP), the committed 9 diterpenoid precursor ( Figure 1 . 4 ). The GGDP precursor is cyclized by taxadiene synthase to produce taxa - 4(5),11(12) - diene, the committed precursor for taxoid biosynthesis ( 67, 68 ) . Paclit axel is produced from taxa - 4(5),11(12) - diene by a series of ~19 - enzymatic steps ( Figure 1 . 4 and Figure 1 . 5 ) ( 60, 69 ) . The exact order of these enzymatic reactions is unclea r ( Figure 1 . 4 ). Numerous taxanes have been identified that could be intermediates in the first half of the pathway (prior to the formation of 10 - deacetylbaccatin III (10 - DAB)), demonstrating the difficulty in defin ing a linear pathway to paclitaxel ( 59, 60, 62 ) . Taxa - 4(5),11(12) - diene is modified by a series of at least 7 cytochrome P450 - dependent hydroxylases, a C4 - C20 - epoxidase, an oxomutase, a C9 oxidase, and 5 acyltransferases ( Figure 1 . 4 , Figure 1 . 5 ) ( 64, 70 - 81 ) . The taxadiene - 5 - - hydroxylates taxa - 4(5),11(12) - diene precursor at the C5 position, and also migrates the double bond to form taxadiene - - ol ( Figure 1 . 4 ) ( 77, 78 ) . Despite great progress identifying taxane structures and screening cDNA libraries from induced Taxus cell cultures, the order of enzyme reactions, and which side - products are produced during the first half of the pathway remain unresolved. In contrast with the first half of the pathway, the biosynthesis of paclitaxel from 10 - DAB is well - described ( Figure 1 . 5 ). The 10 - deacetylbaccatin III 10 - O - acetyltransf erase (DBAT) acetylates 10 - DAB to form baccatin III ( 63, 82, 83 ) . The baccatin III: 3 - amino - 13 - O - phenylpropanoyl CoA transferase (BAPT) acylates baccatin III to add the (3 R ) - - phenylalanyl sidechain ( 71 ) - phenylalanyl CoA ligase responsible for produ cing the activated sidechain intermediate was recently identified, but its substrate specificity was not characterized in depth ( 58 ) - hydroxylase is xylated after the 10 Figure 1 . 5 . Paclitaxel biosynthesis: Part 2. Enzyme abbreviations are as follows: DBAT, 10 - deacetylbaccatin III 10 - O - acetyltransferase, BAPT, baccatin III : 3 - amino - 13 - O - phenylpropanoyl CoA - - phenylalanine CoA ligase, NDTNBT, N - debenzoyltaxol - N - benzoyltransferase. Enzymes marked with (*) are unidentified. Compound abbreviations: 10 - deacetylbaccatin III, (10 - DAB), N - deben zoyl - - deoxytaxol (NDB2T). sidechain is attached to baccatin III ( 80 ) . The last enzyme on the pathway, N - debenzoyltaxol - N - N - debenzoylpaclitaxel to form paclitaxel ( 64, 73 ) . As mentioned, there are hundr eds of taxanes in plant cells derived from taxa - 4(5),11(12) - diene, many of which are either enzyme byproducts or a result of pathway branches ( 21, 60, 84 ) ( Figure 1 . 6 ). Efforts to increase flux toward paclitaxel pr oduction in plant cells have centered on profiling gene expression in methyl jasmonate - induced cell cultures to identify genes involved in the regulation of paclitaxel biosynthesis ( 84 - 86 ) . 11 Figure 1 . 6 . Examples of off - pathway metabolites that divert flux away from paclitaxel production. Genetic transformation techniques with Taxus cells are in development and not yet routine, limiting the engineering capabilities of PCF ( 87 - 89 ) . Using these techniques, several repressive transcription factors of paclitaxel biosynthetic genes were recently identified and may prove useful as targets to increase paclitaxel production in PCF ( 90 ) . 1.1.6 Paclitaxel production Paclitaxel purification fr om the bark of the Pacific yew is low yielding (<0.05 %), environmentally unsustainable, and economically unfeasible ( 37 ) . Even so, paclitaxel was 12 harvested from the yew tree for early studies and clinical trials ( 23 ) . Growing clinical success of paclitaxe l prompted enormous efforts into developing synthetic production methods. However, due to its structural complexity, the complete organic synthesis of paclitaxel is also untenable with more than 40 synthetic steps ( 29 - 34 ) . The discovery of high levels of t he precursors 10 - DAB and baccatin III in the needles of Taxus plants made the development of semi - synthetic methods possible as yew needles are a more renewable resource than yew bark ( 23, 91, 92 ) . The semi - synthetic production of paclitaxel from 10 - DAB wa s developed by the Holton research group in collaboration with BMS ( 36, 93 - 95 ) ( Figure 1 . 7 ). This method substantially improved paclitaxel production compared with isolation from the natural source ( 36 ) . However, m ultiple protection and deprotection steps were required, making this method costly in the long - term. The development of suspended Taxus plant cell fermentation technology to produce biosynthetic paclitaxel is replacing semi - synthesis for commercial product ion ( 37, 96 ) . Phyton Biotech developed this technology and currently supplies a majority of paclitaxel worldwide ( 57, 97 ) . Taxus cells biosynthesize paclitaxel in response to a number of elicitors both organic and inorganic, the strongest and most well - kno wn being methyl jasmonate ( 98 - 100 ) . In the presence of methyl jasmonate, T. baccata PCF produces paclitaxel and baccatin III at levels of ~50 mg/L compared with ~0.4 mg/L in unelicited cells ( 100 ) . However, yields produced from Taxus cell cultures are high ly variable depending on the Taxus sp., the elicitors present, and growth conditions. In addition to paclitaxel production, PCF produces other taxanes as byproducts. Large quantities of 10 - DAB and baccatin III are also produced by PCF and used as semi - synt hetic precursors for the production of clinically - approved docetaxel and 13 Figure 1 . 7 . Semi - synthesis of paclitaxel from 10 - DAB. (i) TESCl, imidazole, DMF, r.t., 96% yield ; (ii) LiHMDS, CH 3 COCl, THF, 82% yield, (iii) LDA, THF, (iv) PMP aldimine, 96% yield for two steps, (v) CAN, CH 3 CN, H 2 O, 88%, (vi) BzCOCl, TEA, DMAP, CH 2 Cl 2 , r.t., 97%, (vii) LiHMDS, THF, - 40 °C, 30 min, (viii) HF/pyridine, pyridine/MeCN, 0 °C to r.t., 18 h, 80% for two steps. The overall yield of paclitaxel is approximately ~50 % without considering the synthesis of the C13 sidechain components. This synthesis is adapted from ( 95, 101 ) . 14 Figure 1 . 8 . Taxanes clinically approved and in clinical trials. A. The natural product paclitaxel. B. Docetaxel. C. Cabazitaxel. D. DJ - 927 (Tesetaxel) completed phase II clinical trials for several cancers. E. BMS - 275183 (Phase I terminated) . F. TL - 00139 (Milataxel) completed phase II clinical trials for malignant mesothelioma. G. MST - 997 (Phase I - terminated). H. TPI - 287 ongoing clinical trials for several cancers and .gov). cabazitaxel, as well as other analogs in clinical trials including TPI - disease ( Figure 4 . 4 ) ( 51 ) . 1.1.7 Semi - biosynthesis of paclitaxel, precursors, and analogs Although PCF methodologies were adapted to produce paclitaxel in commercial quantities, docetaxel, cabazitaxel and other analog s must be semi - synthesized from 10 - DAB or baccatin III, requiring multiple protection/deprotection steps, and large quantities of reagents and organic solvents ( 51, 102 - 109 ) . Paclitaxel production transitioned to PCF because it was more cost - effective and environmentally friendly. Lack of a biosynthetic 15 Figure 1 . 9 . Coupled enzym e biosynthesis of paclitaxel. I. (2 R ,3 S ) - phenylisoserinyl CoA production by PheAT. II. Sidechain attachment to baccatin III by BAPT to produce the docetaxel precursor (R 1 = H) (III). IV. BadA catalyzes the production of benzoyl CoA. V. NDTNBT transfers a b - N of N - debenzoylpaclitaxel to produce paclitaxel. Baccatin III: R 1 = acetyl group. 10 - Deacetylbaccatin III (10 - DAB): R 1 = H. pathway for certain analogs makes in vivo biosynthetic engineering impossible, but development of a heterol ogous pathway in microorganisms could make precursor biosynthesis possible by exploiting the broad substrate specificity of biosynthetic enzymes and feeding precursors into the microorganism growth media. 16 Recently, bioengineering efforts with the paclitaxe l pathway have increased in heterologous organisms such as E. coli , S. cerevisiae , and A. thaliana ( 110 - 120 ) . Thus far, progress is limited to the earliest pathway steps due to incomplete characterization of the pathway ( 111, 119 ) . The eukaryotic, membrane - bound enzymes, such as the cytochrome P450 hydroxylases, must be expressed in yeast or engineered for expression in bacteria ( 81, 111, 121 ) . Byproducts produced by pathway enzymes with uncharacterized substrate specificities ( 78, 122 ) , and unidentified pa thway bottlenecks ( 116 ) , complicate bioengineering attempts. The complexity of the non - linear paclitaxel pathway and its incomplete characterization suggest bioengineering paclitaxel from primary metabolism and minimizing side - product formation will be ext remely difficult ( 60, 96 ) . Given these concerns and the availability of the 10 - DAB and baccatin III precursors from PCF, we proposed the construction of a simple, biocatalytic pathway that bypasses many of the problems associated with engineering the enti re paclitaxel pathway. The approach is versatile, ultimately enabling the production of paclitaxel, analog s, and precursors of interest in E. coli . The cytoplasmic BAPT and NDTNBT Taxus acyltransferases, and two corresponding cognate, bacterial acyl CoA li gases, PheAT and BadA, were combined in a 4 - enzyme biosynthetic reaction ( Figure 1 . 9 ) ( 123, 124 ) . The use of all 4 enzymes with the starting material baccatin III produces paclitaxel. By omitting the NDTNBT acyltra nsferase and the BadA ligase, N - debenzoylpaclitaxel precursors are made. Analogs of pharmaceutical interest are subsequently made by N - acylation. Substituting baccatin III for 10 - DAB leads to the production of docetaxel analogs or the docetaxel precursor, N - debenzoyl - 10 - deacetylpaclitaxel. Benefits of using the late stage paclitaxel biosynthetic enzymes, BAPT and NDTNBT, include bypassing uncharacterized 17 biosynthetic pathway steps and an opportunity to exploit the acyl CoA substrate permissivity and regiose lectivity of these enzymes to make variously acylated analog s of interest. In order to develop this pathway and demonstrate proof - of - principle for paclitaxel and analog biosynthesis, the activities of BadA, a benzoate CoA ligase, and BAPT, a baccatin III: - phenylpropanoyl CoA transferase, were studied ( Figure 1 . 9 ). The substrate specificity of BadA was systematically probed via Michaelis - Menten kinetic characterization with an array of substrates, and activity was also interpreted in the context of six crystal structures ( 125 ) . Targeted mutation of the BadA active site and activity analysis led to new insights regarding enzyme mechanism and expanded substrate specificity compared with wild - type BadA (Chapter 2). T he characterization of BAPT activity in vitro first required protein expression optimization and purification. Initial discovery and characterization of BAPT enzyme activity was limited to estimations of K M with (3 R ) - phenylalanyl CoA and (2 R ,3 S ) - phenylisos erinyl CoA, performed with assays containing impure BAPT lysate ( 71 ) . In this work, BAPT was purified substantially compared with initial experiments, overcoming solubility hurdles (Chapter 3). Determination of the Michaelis - Menten kinetic parameters of BA PT required the synthetic and biosynthetic production of commercially unavailable acyl CoA substrates including (3 R ) - phenylalanyl CoA and (2 R ,3 S ) - phenylisoserinyl CoA. BAPT was combined with PheAT to demonstrate the biosynthesis of paclitaxel or docetaxel precursors from (2 R ,3 S ) - phenylisoserine and baccatin III or 10 - DAB, respectively (Chapter 4). 18 Ultimately, BAPT was combined with PheAT, NDTBT, and BadA to produce N - 2 - furanyl - N - debenzoylpaclitaxel, a biologically active analog of paclitaxel (Chapter 4). Th is demonstrates the potential for these enzymes to be combined and developed in bacteria to produce paclitaxel/docetaxel/cabazitaxel precursors and other analog s of clinical interest. Semi - biosynthesis of docetaxel (or precursors) by this method has the ab ility to shorten the number of synthetic steps, potentially increasing production and lowering the cost to patients. By exploiting the substrate specificities of enzymes in this pathway the number of steps required to produce a given paclitaxel analog may be reduced. The combination of bacterial CoA ligases and plant acyltransferases to produce acylated natural products may be extended to additional analogs by pairing an acyltransferase with acyl CoA ligases with different substrate specificities. Organic synthesis of biologically relevant, structurally complex natural products is often difficult and costly, requiring large volumes of environmentally harmful organic solvents. 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(2015) Kinetically - and crystallographically - guided mutations of a benzoate CoA 31 ligase (BadA) elucidate mechanism and expand substrate permissivity, Biochem istry - US 54 , 6230 - 6242. 32 Chapter 2 is adapted from the following publication: Kinetically and crystallographically guided mutations of a benzoate CoA ligase (BadA) elucidate mechanism and expand substrate permissivity. Biochemistry - US , 54 , 6230 - 62 42 , 2015. Chelsea K. Thornburg, Susa n Wortas - Strom, Meisam Nosrati, James H. Geiger and Kevin D. Walker BadA crystal screening and structure determination was performed by Susan W. Strom and Meisam Nosrati. All other work was performed by Chelsea K. Thor nburg. 33 Chapter 2. Kinetically - and crystallographically - guided mutations of a benzoate CoA ligase (BadA) elucidate mechanism and expand substrate permissivity 2.1 Introduction Acyl - group adenylation by ATP, in both primary and specialized metabolism, is common to all living organisms. The superfamily of ATP - dependent adenylation enzymes (PFAM00501) is organized into three classes ( 1 ) . Class I includes the non - ribosomal peptide synthetase (NRPS) adenylation domains, the acyl CoA ligases, luciferase oxidoreductases ( 2 ) . Classes II and III comprise the aminoacyl - tRNA synthetases and NRPS - independent siderophore (NIS) enzymes, respectively ( 3 ) . The focus of the current study centers on an acyl CoA ligase from Class I. Plants and bacteria employ aroyl CoA thioesters for the biosynthesis of specialized metabolites. For example, anthraniloyl CoA is a precursor of a quorum sensing molecule in Pseudomonas aeruginosa ( 4 ) ; benzoyl CoA lies on the pathway to enterocin (a bacteriocin) in the marine actinomycete Salinispora ( 5 ) , to t he chemotherapeutic Taxol in Taxus plants ( 6, 7 ) , and to the volatile fragrant benzylbenzoate in plants ( 8 ) . Biosynthesis of the anti - bacterial and anti - fungal aureothin proceeds through 4 - nitrobenzoyl CoA in soil bacterium Streptomyces thioluteus ( Figure 2 . 1 ) ( 9, 10 ) . Accounts of aerobic and anaerobic degradation of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) to benzoate derivatives in bacteria inclu de the characterization of several different aroyl CoA ligases from Rhodopseudomonas palustris , Burkholderia xenoverans , Pseudomonas strain 34 Benzoyl CoA Enterocin Taxol Benzylbenzoate Anthraniloyl CoA 4 - Nitrobenzoyl CoA PQS ( Pseudomonas Quorum Signal) Aureothin Figure 2 . 1 . Representative natural products derived from acyl CoA thioesters. 35 CBS3, Thauera aromatica , and Pseudomonas aeruginosa ( 11 - 16 ) . R edox enzymes in these bacteria target the aroyl CoA thioesters for dearomatization and ring cleavage along catabolic pathways ( 17, 18 ) . These organisms are thus candidates for bioremediation of man - made PAHs and PCBs to reduce their environmental impact ( 1 7, 19 ) . ATP - dependent CoA ligases use two half - reactions to catalyze thioesterification ( Figure 2 . 2 ) ( 20 ) . The enzyme binds an alkyl - or aryl carboxylate and a Mg 2+ - ATP complex for subsequent coupling during the fi rst half - reaction. The oxyanion of the carboxylate nucleophilically attacks ATP, releases diphosphate, and forms an acyl - AMP mixed anhydride intermediate. In the second half - reaction, the nucleophilic thiol of CoA attacks the carbonyl of the acyl - AMP inter mediate to make the thioester. Other crystal structures from this ligase family support the hypothesis that the enzymes adopt adenylation - and thiolation - conformations to catalyze their respective half - reactions ( 15, 21, 22 ) . Several active aroyl CoA ligas e structures have been determined in complex with acyl carboxylates, cofactors, acyl - adenylates, AMP, ATP, and CoA ( 14, 15, 21, 23 - 29 ) . R. palustris , chosen as a potential bacterial candidate for bioremediation, has at least three distinct aroyl CoA ligase s with different substrate specificities and regulatory characteristics ( 13, 30 ) . Of these, a benzoate CoA ligase (designated BadA) was identified in an earlier study ( 12 ) that reported on the relative V max (i.e., V rel ) values of BadA for different substra tes compared to benzoate ( 12 ) . Here, we explicitly calculated the apparent kinetic parameters ( and ) of BadA for 25 different carboxylate substrates to further understand the steric, mesomeric, and inductive effects of the substituents on the mechanism. We intend to repurpose BadA for use in coupled enzyme assays to 36 Figure 2 . 2 . ATP - dependent, two - step mechanism of a benzoate: CoA ligase in the presence o f magnesium. BzO: Benzoate; Bz - AMP: Benzoyl AMP; and BzCoA: Benzoyl Coenzyme A. The ortho - , meta - and para - carbons of the aryl ring of BzO are highlighted with carbon numbering 2 - , 3 - , and 4 - , respectively. biosynthesize non - natural acyl CoAs. These thio esters will serve as substrates for CoA - dependent N - and O - acyltransferases that acylate many bioactive specialized metabolites from plants ( 31, 32 ) . Calculation of the intrinsic catalytic constants of BadA for non - natural carboxylates is an important step towards this goal. Also described herein are five X - ray crystal structures of recombinant BadA at ~1.80 Å resolution complexed with various aryl carboxylates and one with benzoyl - AMP (Bz - AMP). BadA is structurally similar to other CoA ligases in its class , comprising separate N - terminal and C - terminal domains joined by a flexible hinge. Based upon the structures of other aroyl CoA ligases, we expected the C - terminal domain of BadA - carboxylate complexes to remain in an "un - rotated" adenylate - forming conform ation as seen in other benzoate derivatives in complex with ligases ( 21, 23, 27 ) . Surprisingly, however, each complex showed BadA in the conformation primed for thioesterification. The homologous human medium chain fatty acyl CoA synthetase (ACSM2A) ( 24 ) p rovides precedent for the binding of benzoate (BzO) to BadA in the thiolation conformation. By analogy, ACSM2A co - crystallized with the surrogate substrate ibuprofen in the thiolation conformation ( 24 ) . In addition, each of the aryl carboxylates binds the BadA active site in a unique configuration rotated ~60° about the phenyl ring relative to BzO bound in other 3 7 structures and the Bz - AMP bound in BadA. Here, mutagenesis of the active site supports the importance of both orientations of the substrate in the active site. Together, the data support a mechanism where benzoate tightly binds BadA in the thiolation conformation, followed by rotation in the active site upon ATP binding for the acyl adenylation step. The structures further provide a basis for rationa l mutagenesis to remove sterically challenging residues from the active site and expand the substrate scope of BadA. The results of a series of mutations are also described here. Using these structures, we also determined that the non - conserved Lys427, pre sent in the BadA active site in the thiolation conformation, is not required for acyl adenylate formation but is necessary for thioesterification. 38 2.2 Experimental 2.2.1 Materials Benzoic acid (98%) was obtained from Mallinkrodt Pharmaceuticals (St. Louis, MO). 2 - Aminobenzoic acid (98%), 3 - aminobenzoic acid (98%), 2 - cyanobenzoic acid (96%), cyclohexane carboxylic acid (98%), 1 - cyclohexene - 1 - carboxylic acid (97%), 3 - cyclohexene - 1 - carboxylic acid (98%), 4 - ethylbenzoic acid (99%), 2 - hydroxybenzoic acid (99+%), 3 - hydro xybenzoic acid (99%), 2 - methoxybenzoic acid (98%), 2 - nitrobenzoic acid (95%), 3 - nitrobenzoic acid (99%), 2 - picolinic acid (99%), and thiophene - 2 - carboxylic acid (99%) were purchased from Alfa Aesar (Ward Hill, MA); acetyl CoA (98%). 4 - Aminobenzoic acid (99 %), ATP (98%), benzoyl CoA (98%), 2,6 - dichlorobenzoic acid (98%), 3 - chlorobenzoic acid (99%), cinnamic acid (99%), 3 - cyanobenzoic acid (98%), 4 - cyanobenzoic acid (98%), 3,4 - dimethylbenzoic acid (98%), 2 - fluorobenzoic acid (97%), 3 - fluorobenzoic acid (97%), 4 - fluorobenzoic acid (98%), L - histidine (99%), 4 - hydroxybenzoic acid (99%), 2 - methylbenzoic acid (99%), 3 - methoxybenzoic acid (99%), 4 - methoxybenzoic acid (99%), 3 - methylbenzoic acid (99%), 4 - methylbenzoic acid (98%), phenylacetic acid (99%), L - phenylalan ine, and L - tyrosine (98%) were obtained from Sigma - Aldrich (St. Louis, MO). 2 - Chlorobenzoic acid (>98.0%) and 4 - chlorobenzoic acid (99%) were from TCI America (Portland, OR). 4 - Nitrobenzoic acid (99%) was purchased from Acros Organics (New Jersey). Coenzym e A (~95%) was purchased from Lee Biosolutions (St. Louis, MO). 39 2.2.2 Plasmids The badA cDNA from Rhodopseudomonas palustris was generously provided by Caroline Harwood (University of Washington, Seattle, WA). The gene was amplified by PCR and subcloned into th e pET28a (Novagen) expression vector with the following forward primers: 5' - TAT GAA TGC AGC CGC GGT C - 3', 5' - TGA ATG CAG CCG CGG TCA C - 3' and reverse primers: 5' - GTC AGC CCA ACA CAC CC - 5', and 5' - TCG AGT CAG CCC AAC ACA CC - 3'. Directional cloning of badA w as confirmed by DNA sequencing (Michigan State University Genomics Core). The resulting plasmid was named pBadA. 2.2.3 BadA protein expression and purification The pBadA expression vector (pET28a) was used to transform BL21(DE3) E. coli (Invitrogen, Carlsbad, CA ). A single colony was selected and used to inoculate a 10 - mL culture of Luria - Bertani (LB) media (Accumedia) containing 50 µg/mL kanamycin (Roche). The culture was grown overnight at 37 °C. This seed culture (5 mL) was used to inoculate fresh LB media (1 L) containing 50 µg/mL kanamycin. This culture was grown at 37 °C until A 600 = 0.8. Gene expression was induced by 0.5 mM - D - 1 - thiogalactopyranoside (IPTG), and the cultures were grown for 5 h at 18 °C and harvested by centrifugation at 6,000 g . The bacterial pellet was resuspended in Buffer A (50 mM Na 2 PO 4 , 300 mM NaCl, 15 mM imidazole, 5% glycerol, pH 8.0) containing EDTA - free Protease Inhibitor Cocktail tablets (Roche Life Sciences, Indianapolis, IN). Cells were lysed with a Misonix XL 2020 son icator and centrifuged at 18,000 g for 30 min. The supernatant was passed through a 0.2 µm filter (Millipore, Billerica, MA) and loaded onto a Ni 2+ NTA Qiagen column pre - equilibrated with Buffer A. The column was washed with 5 column volumes (CV) of Buffer A and eluted with 3 CV of Buffer A containing 250 mM 40 imidazole. Each fraction was analyzed by SDS - PAGE. Fractions containing BadA were combined, loaded into a 10 kDa MW cutoff Dialysis Cassette (Thermo Scientific Pierce, Grand Island, NY), and dialyzed ove rnight against Buffer B (50 mM NaPO 4 containing 5% glycerol, pH 8.0) for kinetic analysis or Buffer C (20 mM Tris, pH 8.0) for protein crystallization experiments. Dialyzed protein was concentrated in a Millipore Amicon Ultra 30 kDa cutoff concentrator to ~10 mg/mL (estimated by the Coomassie (Bradford) Protein Assays (Thermo Scientific Pierce, Grand Island, NY). The molecular weight of BadA (501 amino acids) was verified by liquid chromatography/electrospray ionization/ mass spectrometry (LC - ESI/MS) on a Q - ToF Ultima Global mass spectrometer (Waters, Milford, MA). Protein aliquots were frozen in liquid nitrogen and stored at 80 °C. 2.2.4 BadA kinetic assays Stocks of ATP (10 mM) and CoA (10 mM) were dissolved in Buffer B, MgCl 2 (at 100 mM) was stored in water, a nd stocks of the aromatic carboxylic acids (each at 100 mM) were dissolved in methanol. Aromatic carboxylic acids were then diluted in water. To establish steady - state kinetic rates of BadA with respect to protein concentration and time for each carboxylat e (1 µM to 4 mM) and other reactants: ATP (250 µM), CoA (250 µM), MgCl 2 (750 µM) were combined in Buffer B and pre - incubated at 31 °C for 10 min before the addition of 0.1 to 20 µg/mL BadA (90 µL total volume). Assays were acid - quenched (pH 3) with 8.8% fo rmic acid in water. Acetyl CoA was added as an internal standard at a final concentration of 1 µM. Methanol concentrations were held constant at 1% (v/v) among assays with varied concentrations of carboxylic acids. 41 2.2.5 BadA assay analysis by liquid chromatogr aphy mass spectrometry Liquid chromatography electrospray tandem mass spectrometry (LC ESI/MS/MS) in negative ion mode was used to quantify the biosynthetic acyl CoA products. An autosampler (at 10 °C) connected to a UPLC system (Waters Corp., Milford, MA) injected a 10 - µL aliquot of each assay onto an Ascentis Express C18 HPLC column (2.7 µm, 5 cm × 2.1 mm, at 30 °C, Sigma - Aldrich). The column was eluted at 0.4 mL/min with 2.5% solvent B (100% acetonitrile) and 97.5% solvent A (0.05% triethylamine in water (pH 5.5)) with a 0.5 min hold, followed by a linear gradient to 20% solvent B over 4 min, then increased to 100% solvent B over 2 min, and finally lowered to 2.5% solvent B over 0.5 min. The needle was washed with 2 mL each of 100% isopropanol and then wi th 10% acetonitrile in water prior to each injection. The HPLC effluent was directed to an electrospray ionization mass spectrometer (Quattro Premier XL, Waters Corp, Milford, MA) in negative ion mode, with a cone voltage of 60 V and collision energy of 44 eV. Each aryl CoA was quantified by multiple reaction monitoring of the [M H] m / z 408 transition, common to each aryl CoA tested ( Figure 2 . 3 ). Peak areas (calculated using the MassLynx data analysis software, Waters Corp., Milford, MA) were converted to product concentrations using a stand ard curve for a series of benzoyl CoA concentrations (61 nM to 15.6 µM, n = 3) that were normalized to an internal standard acetyl CoA (1 µM). Figure 2 . 3 . Acyl CoA detectio n by tandem mass spectrometry. Multiple - reaction monitoring (MRM) LC - ESI/MS/MS [M - H] m / z 408 transition ion in negative ion mode for aryl CoA thioesters. 42 2.2.6 BadA mutations Point mutations of the badA gene were generated by site - directed mutagenesis, using the following forward and reverse primer pairs (mutated bases are underlined): A227G : 5' - CCA AAC TGT TTT TCG GC T ACG GCC TCG GCA ACG - 3' and 5' - CGT TGC CGA GGC CGT A GC CGA AAA ACA GTT TGG - 3'; L333A : 5' - CGG CTC GAC CGA GAT G GC G CA CAT CTT TCT GTC GAA C - 3' and 5' - GTT CGA CAG AAA GAT GTG CGC CAT CTC GGT CGA GCC G - 3'; H334A : 5' - CGG CTC GAC CGA G AT GCT G GC G AT CTT CCT GTC GAA TTT G - 3' and 5' - CAA ATT CGA CAG GAA GAT CGC CAG CAT CTC GGT CGA GCC G - 3'; I335A : 5' - CCG AGA TGC TGC AC GCG TTT CTG TCG AAC CTG C - 3' and 5' - GCA GGT TCG ACA GAA A CG C GT GCA GCA TCT CGG - 3'; K427A : 5' - CGA CAT GCT GGC GGT CAG CGG C AT CTA TGT CAG CCC GTT CGA GAT CG - 3' and 5' - GCC GCT GAC CGC CAG CAT GTC GTC GGT GCG GCC CGC ATA GGT GTA G - 3'. The pBadA plasmid was PCR amplified using Phusion HF polymerase (New England Biolabs, Ipswich, MA) under the following protocol: 95 °C for 3 min, fol lowed by 20 cycles of 95 °C for 1 min, 58 °C for 1 min, 72 °C for 4.5 min, with a final elongation step of 72 °C for 7 min. PCR product was digested with DpnI (New E. coli (Invitrogen, Thermo L ife Sciences, Grand Island, NY). The resulting colonies were inoculated into starter cultures and grown for DNA purification (PureYield Plasmid MiniPrep System, Promega, Fitchburg, WI) and DNA sequencing for the badA point mutations at the Michigan State U niversity Research Technology Support Facility. Mutant plasmids were used to transform BL21(DE3) E. coli (Invitrogen). The expression, isolation and purification of mutant enzyme were identical to that described for wild - type BadA. 43 2.2.7 Kinetic analysis To calc ulate the kinetic constants, each substrate was varied (1 assays. The steady - state concentrations of assay reagents were 250 µM ATP, 250 µM CoA, 2.5 mM MgCl2, 0.1% methanol in 50 mM NaHPO 4 buffer (pH 8) with varying concentration of carboxylate substrate. BadA concentrations are noted for each substrate. Resultant CoA thioester products were quantified after terminating the reaction as described previously. The apparent kinetic parameters ( and ) were calculated by non - linear regression with Origin Pro 9.0 software (Northampton, MA) (Figures A .1. 1 A .1. 24), using the Michaelis - Menten equation: v o = /( + [S]) . Relative steady - state rates for mutant enzymes Ala227Gly - BadA, Leu332Ala - BadA, His333Ala - BadA, and Ile334Ala - BadA were calculated for 15 aryl carboxylates. Assays with purified 2+ reported as percentages relative to BadA with benzoate. For the Lys427A - BadA mutant, the biosynthetic benzoyl - AMP and benzoyl CoA products catalyzed by the mutant were analyzed separately by HPLC (with A 254 detection) and LC - ESI - MS/MS. Control assays conta ined the necessary cofactors and reagents but no mutant enzyme. 2.2.8 BadA crystal structures 2.2.8.1 Crystallization of R . palustris benzoate: coenzyme A ligase (BadA) Purified BadA protein at (17.5 mg/mL) was used for de novo protein crystallization trials using six different 96 - condition Crystal Screen (Hampton Research) matrices. A Gryphon Protein Crystallization robot (Art Robbins Instruments) was used for setting up sitting - drop 44 vapor diffusion crystallization trials. The trials were kept at 22 °C throughout crys tallization. About 45 different conditions produced crystals of varying quality; the best was from the Wizard I/II Screen A10 condition (20% PEG - 2000 MME (w/v), 0.1 M Tris - HCl, pH 7). This condition was then optimized in crystal boxes employing the hanging - drop vapor diffusion crystallization method. 2 µ L hanging drops were used consisting of an equal measure of protein and reservoir solutions. The best crystals, used for data collection, were grown from a reservoir solution containing 0.1 M Tris - HCl, pH 7 .0 and 15% PEG 3350 (w/v). 2.2.8.2 Co - crystallization to obtain the ligand bound structure Co - crystallization was employed by adding separately each carboxylic acid ligand (4 equiv) to the BadA protein solubilized in 0.1 M Tris buffer, pH 8.0. For the multiple - com ponent cocrystallization, ATP, CoA and benzoic acid (each at 1 eq) were added to the protein solution. Each co - crystallization mixture was incubated for 10 min at 4 °C followed by crystallization using a reservoir of 20 mM Tris buffer at 15% PEG 3350 at va riable pH. The best crystals grew in a range of pH from 6.5 to 7.5. 2.2.8.3 Data processing and refinement of BadA Crystals were soaked in cryoprotectant (0.2 M Tris - HCl, pH 6.5 7.5, containing 20% PEG - 3350 and 20% glycerol), mounted in CryoLoops (Hampton Resear ch), and then flash - frozen in liquid nitrogen. The native X - ray diffraction intensity data were collecte d under a continuous stream of nitrogen at 100 K on beamline 21 - ID - G, LS - CAT (Argonne National Laboratory, Advanced Photon Source, Chicago, IL) at a wav elength of 0.97872 Å. Raw diffraction data were indexed using the HKL2000 software package ( 33 ) . A search of the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank 45 (PDB) revealed one known structure for a benzoate CoA ligase (BCL M ) from Burkholderia xenovorans LB400 (PDB 2V7B) ( 14 ) . Query of the Swiss - Model server ( 34 ) produced a threaded protein structure homology - model of BadA based on the BCL M structure. The BadA structure was solved by molecular replacement using this model an d the MOLREP program in the CCP4 suite ( 35 - 37 ) . Though the N - terminal domain was well - positioned within the density, the C - terminal domain was not. The model was corrected using the Buccaneer program in the CCP4 suite to thread the amino acid sequence into the density. Further corrections were made manually using the COOT program ( 38 ) . Jligand version 1.0.9 ( 39 ) of the CCP4 suite was used to add the template restraints for the ligands prior to refinement. The structures were refined using REFMAC in the CCP4 program suite. Water molecules were added using REFMAC and COOT near the end of the refinement. 2.2.9 Calculation of covalent van der Waals volumes and lengths The molecular volumes of the substituents on the aryl carboxylate substrates were estimated as Conn olly solvent - excluded volume ( 40 ) using ChemBio3D Ultra software (ver. 13.0, Perkin Elmer) and a probe radius of 0.1 Å. The volume of a phenyl anion was subtracted from the estimated volume of a phenyl attached to a substituent. The geometries of polyatomi c substituents such as CH 3 , OH, MeO, CN, NH 2 , and NO 2 , attached to phenyl were MM2 energy minimized using the default parameters of the ChemBio3D Ultra software. The covalent van der Waals lengths of CN and Cl substituents attached to a phenyl ring were ca lculated using the covalent bond distance between two atoms and atomic radius of the terminal atom. 46 2.3 R esults 2.3.1 Solving the BadA s tructure BadA crystallized as a complex with benzoate (BzO) in the absence of added benzoate. BadA also co - crystallized with 2 - fluorobenzoate (2 - F - BzO), 2 - methylbenzoate (2 - Me - BzO), 3 - furoate (3 - Fur), and thiophene - 2 - carboxylate (Thio - 2 - C) ( Figure 2 . 2 and Figures A1 A24 for structures). BadA was also co - crystallized with both benzoate an d ATP resulting in a bound Bz - AMP intermediate, the product of the first half - reaction (see Figure 2 . 2 ), bound in the active site. All structures were refined to resolutions of at least 1.8 Å (Table A1) and helped guide the interpretation of the kinetics data. Figure 2 . 4 . Crystal structure of BadA in complex with benzoate . The substrate is shown in yell ow and en closed in electron density map ( 2F o - F c ). The N - termin al domain (green) and the C - terminal domain (blue) in the "thiolation conformation." BadA (PDB: 4EAT) 2.3.1.1 Domain o rientation The BadA structures in complex with BzO (PDB: 4EAT) ( Figure 2 . 4 and Figure A .1. 26), 2 - Me - BzO (PDB: 4RLQ), Thio - 2 - C (PDB: 4RMN), 3 - Fur (PDB: 4RM3), 2 - F - BzO (PDB: 4RM2) and Bz - AMP (PDB: 4ZJZ) (Figure A .1. 26) had the characteristic N - terminal and 47 C - terminal domains of other ATP - dependent adenylases ( 2, 28 ) . The N - terminal (residues 1 434) and C - te rminal (residues 435 522) domains of BadA fold analogously to the benzoate CoA ligase BCL M (PDB: 2V7B) ( 14 ) with an RMSD between the peptide backbones of ~0.5 Å and 0.6 Å, respectively ( 41 ) . The N - terminal domain contains the benzoate binding site, and t he C - terminal domain contacts the outer edge of the benzoate binding pocket, positioning benzoate in the active site through a charged interaction between the carboxylate of benzoate and Lys427 ( Figure 2 . 4 ). 2.3.1.2 Featur es of the BadA active site The BadA active site is largely hydrophobic with many binding contacts between the substrate and the polypeptide backbone. In all BadA structures bound to the aryl carboxylate, the active site coordinates the carboxylate substrat e with Lys427. In this position, the para - carbon of BzO neighbors the carbonyl of Leu332 along the peptide backbone, and the two meta - carbons of BzO point towards His333 and Ala227 on either side ( Figure 2 . 5 , A and D). The si - and re - faces of the BzO are positioned between backbone amide bonds comprising Gly327, Ser328, and Thr329 on one face, and Tyr228 on the other face. Co - crystallization of BadA with BzO and ATP yielded a Bz - AMP intermediate in the active site with the C - terminal domain also poised in the thiolation conformation ( 21, 26 ) . The active sites of structures bound to BzO or Bz - AMP showed almost no structural differences. However, when Bz - AMP forms, Lys427 moves from an interaction with the carboxylate of the substrate to several polar contacts with the Bz - AMP and the peptide backbone ( Figure 2 . 5 , B and D). We evaluated the importance of Lys427 of BadA for catalysis by making a Lys427Ala - BadA mutant and compared its activity against BadA. 48 The mutant made benzoyl CoA at a rate = (83 ± 1.8 ) × 10 - 5 s 1 ), more than 33000 - fold slower than wild type BadA ( = 28 s 1 ), yet the (1.5 ± 0.31 µ M) for this mutant was nearly the same for the w ild - type BadA ( 4.4 ± 0.65 µM ) ( Figure A .1. 27). The mutant and BadA catalyzed approximately equal amounts of benzoyl - AMP in the absence of CoA (Figure A .1. 28). These data indicated that Lys427 is needed for the thiolation reaction. When the Bz - AMP anhydride forms, other structural differences include a decrease in the dihedral angle between the carboxylate and the aryl ring from 37° for BzO to 12° for the anhydride. Further, Tyr432 shifted toward the phosphate oxygen of the AMP anhydride and away from Arg421 to accommodate the Bz - AMP (Figure A .1. 29). The benzoyl moiety also rotates ~60° about the C 6 - axis of the aromatic ring and moves the ortho - carbon from an unhindered region of the active site towards residue Ala227, which previously resided near the meta - c arbon ( Figure 2 . 5 , C and D). This rotation positions the benzoyl moiety of Bz - AMP similar to those of benzoates bound in other CoA ligase structures, regardless of the C - terminal domain orientation ( 14, 21, 23 ) . 49 A B C D Figure 2 . 5 . The active site of the BadA structure. A. Three - dimensional rendering of BadA in complex with benzoate (BzO) (cyan) (PDB: 4EAT) and B. benzoyl - AMP (Bz - AMP) (yellow) (PDB: 4ZJZ); key, steric and substrate binding residues (green) are highlighted. C. Overlay of the active site orientations of BzO (cyan) and Bz - AMP (yellow) in complex with BadA. Structure alignments were made with UCSF Chimera 1.10 ( 42 ) . D. Two - dimensional rendering of the active site showing distances (Å) between BzO ( bold black structure ) and Bz group of Bz - AMP ( bold blue structure ) and key, steric active site residues. ( Insets ) Two views of the dihedral angle between the carboxyl ate oxygens and the phenyl ring of BzO and Bz - AMP (partial structure) bound in BadA. Heteroatoms are colored as oxygen (red), phosphorus (orange), and nitrogen (blue). 50 2.3.2 Kinetic properties of BadA The R. palustris BadA was recombinantly overexpressed in E . coli and incubated separately with various heteroaromatic, alkenoyl, alkyl, and aryl carboxylates to calculate the kinetic constants for each substrate ( Table 2 . 1 ). Benzoate substrates with substituents of differi ng steric and electronic properties were chosen to systematically probe the active site of BadA. We quantified the biosynthetic CoA thioester products by LC - ESI - MS/MS. 2.3.3 Substrate t urnover by BadA The (28 ± 0.9 s 1 ) and (4.4 ± 0.65 µM) of BadA for the natural substrate BzO are consistent with those reported earlier ( = 27 s 1 ; = 0.6 2 µM) ( 12 ) . These values were compared to the Michaelis parameters of BadA for 21 mono - substituted benzoates, three cyclohexane deriv ed carboxylates, and two heteroaromatic carboxylates to their corresponding CoA thioesters ( Table 2 . 1 ). Although K M is not a true dissociation constant, in this study it will serve as a means of comparing enzyme in teractions with different carboxylate substrates. 2.3.3.1 Halogenated b enzoates BadA turned over substrates 2 - F - , 3 - F - , and 4 - F - BzO as well as BzO, and the values of BadA for 2 - F - BzO and 4 - F - BzO were similar to that for BzO, but 3 - F - BzO was an outlier ( Table 2 . 1 ). Interestingly, the for 3 - F - BzO was 17 - fold higher than for BzO, reducing its relative catalytic efficiency 13 - fold and suggesting that an interaction between the meta - position and active site r esidues affected binding but not turnover. 51 Table 2 . 1 . Kinetic p arameters of BadA for v arious s ubstrates. Substrate a (s - 1 ) (µM) / (s - 1 µM - 1 ) BzO 28 ± 0.9 4.4 ± 0.65 6.4 Thio - 2 - C 4.8 ± 0.2 37 ± 5.7 0.13 3 - Fur 6.7 ± 0.5 71 ± 12 0.094 PhAc ND - - Cinn ND - - Non - aromatic carboxylates 3 - Cyc 16 ± 1.03 56 ± 9.1 0.29 1 - Cyc 5.6 ± 0.24 52 ± 7.1 0.11 Cyc 13.5 ± 1.07 1470 ± 213 0.0092 Ortho - substituted benzoates 2 - F 3 4 ± 0.7 8.1 ± 0.91 4.2 2 - OH 0.63 ± 0.01 1.6 ± 0.2 0.39 2 - NH2 3.9 ± 0.12 21 ± 2.3 0.19 2 - CN 0.11 ± 0.0034 213 ± 22 0.00052 2 - Cl 2.2 ± 0.07 130 ± 12 0.017 2 - Me 1.2 ± 0.042 44 ± 6.1 0.027 2 - MeO 0.011 ± 0.0005 789 ± 82 0.000014 2 - NO2 0.27 ± 0.011 2015 ± 167 0.00013 Meta - substituted benzoates 3 - F 35 ± 1.4 73 ± 11 0.48 3 - OH 1.8 ± 0.04 229 ± 18 0.0079 3 - NH2 1.8 ± 0.11 174 ± 32 0.010 3 - CN Slow - - 3 - Cl 0.33 ± 0.01 290 ± 23 0.0011 3 - Me 0.54 ± 0.018 215 ± 26 0.0025 3 - MeO ND - - 3 - NO2 ND - - Para - subs tituted benzoates 4 - F 22 ± 0.66 6.6 ± 0.9 3.3 4 - OH 0.93 ± 0.037 158 ± 17 0.0059 4 - NH2 0.18 ± 0.004 25 ± 3 0.0072 4 - CN Slow - - 4 - Cl 0.29 ± 0.012 83 ± 11 0.0035 4 - Me 0.7 ± 0.035 590 ± 73 0.0012 4 - MeO ND - - 4 - NO2 ND - - ND: Not Detectable, below de tection limit. Slow: The CoA thioester p roduct was detected at 0.4 pmol s - 1 at apparent saturation, even at BadA concentrations up to 100 ug/mL. Initial steady - state rates at low substrate concentrations were too slow to calculate kinetic parameters. Stand ard error in parentheses. a Thio - 2 - C: thiophene - 2 - carboxylate, 3 - Fur: 3 - furoate, PhAc: phenylacetate, Cinn: cinnamate, 3 - Cyc: 3 - cyclohexene - carboxlyate, 1 - Cyc: 1 - cyclohexene - carboxlyate, Cyc: cyclohexane carboxylate. Aromatic ring positions are designated a s o - , m - , and p - ( ortho - , meta - , and para - , respectively). Standard errors in parentheses were calculated for the non - linear regression fit by the Origin Pro 9.0 software. 52 BadA turned over 2 - chlorobenzoate (2 - Cl - BzO) better than 3 - Cl - BzO or 4 - Cl - BzO, i n a trend similar to the fluorobenzoates. However, compared to BzO, turnover rates were ~10 - fold slower for 2 - Cl - BzO and 100 - fold slower for both 3 - Cl - and 4 - Cl - BzO. The values of BadA for the Cl - r than the corresponding F - / values of BadA for the Cl - BzO substrates were considerably reduced (~380 - fold for 2 - Cl - BzO and up to ~6000 - fold for 3 - Cl - BzO) compared to that for BzO. These data su ggest the larger chlorine ( 43 ) likely affects the binding interaction of all regioisomers of the chlorinated substrates and prevents them from regularly adopting a catalytic conformation, causing slower turnover ( Table 2 . 1 ). 2.3.3.2 Benzoates with strongly electron - withdrawing substituents The 3 - NO 2 - and 4 - NO 2 - BzO substrates were not productive, while 2 - NO 2 - BzO was turned over ( Table 2 . 1 ). Similarly, 2 - cyanobenzoate (2 - CN - BzO) was t he only productive substrate among the isomeric CN - BzO substrates, but ~2.5 - fold less active than 2 - NO 2 - BzO ( Table 2 . 1 ). The of BadA for 2 - CN - BzO was ~10 - fold lower than for 2 - NO 2 - BzO (2000 µM). These dat a are consistent with the observation that substituent regiochemistry affects substrate binding and subsequent turnover. 2.3.3.3 Benzoates with strongly electron - donating substituents The catalytic efficiency of 2 - hydroxybenzoate (2 - OH - BzO) was 16 - fold lower than BzO, due primarily to its lower ( Table 2 . 1 ). By comparison, the values of BadA for 3 - OH - and 4 - OH - BzO) were substantially higher than for 2 - OH - BzO, yet were turned over similarly. 53 BadA did not bind 2 - aminobenzoate (2 - NH 2 - BzO) ( = 21 µM) as well as 2 - OH - BzO (1.6 µM), and was turned over 6 - fold faster than 2 - OH - Bz ( Table 2 . 1 ). The catalytic efficiencies of BadA for 3 - NH 2 - BzO and 3 - OH - BzO were sim ilar (~0.1 s 1 ·µM - 1 ) because of their comparable and values. 4 - NH 2 - BzO binds BadA ~6 - fold with more apparent affinity ( - OH - BzO ( to the ~6 - fold difference in product release (0.18 and 0.93 s 1 , respectively) of the corresponding CoA thioesters, thus equalizing the catalytic efficiencies of BadA for these substrates. BadA turned over each Me - BzO regioisomer in almost the same order of reactivity as OH - , NH 2 - , and Cl - su bstituted substrates ( ortho > meta > para ) ( Table 2 . 1 ). The values for BadA with Me - BzO substrates compared to BzO were slower, and the values were higher. The larger Michaelis constants were the dominant factors that affected the catalytic efficiency of BadA for the Me - substituted substrates. For the MeO - BzO substrates, BadA turned over the 2 - MeO - BzO 2,000 - fold slower than BzO, with a 180 - fold higher than for BzO. 3 - Methoxybenzoyl (3 - MeO - Bz) CoA and 4 - MeO - Bz CoA were not detectable under standard assay conditions, and were below the detection limit (at 0.5 pmol/mL) of the mass spectrometer. 2.3.3.4 Turnover of heteroaromatic carboxylates BadA turned over the two heteroaromatics Thio - 2 - C and 3 - Fur at similar rates ( Table 2 . 1 ). The and the of BadA for Thio - 2 - C are ~6 - fold slower and ~8 - fold higher, respectively, than the values for BzO; whereas, the values for 3 - Fur were ~4 - fold sl ower and ~14 - fold higher, respectively. These data showed that 3 - Fur was turned over (1.4 - fold) 54 faster by BadA, but had less apparent affinity (1.9 - fold) for the enzyme than Thio - 2 - C, thus equalizing their catalytic efficiencies at ~0.1 s - 1 ·µM - 1 ( Table 2 . 1 ). 2.3.3.5 Turnover of non - aromatic carbocycle carboxylates Intrinsic values of BadA for non - aromatic carboxylates are reported here for the first time. The turnover rates of cyclohexane carboxylate, 1 - cyclohex ene - 1 - carboxylate (1 - Cyc), and 3 - cyclohexene - 1 - carboxylate (3 - Cyc) were similar (only 5 to 1.8 - fold lower) to that of BzO. The of cyclohexane carboxylate (Cyc) was ~330 - fold lower than BzO among these carbocycles. This trend is also reflected in the lower catalytic efficiency of BadA for Cyc than for 1 - Cyc and 3 - Cyc. However, the of BadA for Cyc was surprisingly 48% compared with the turnover of BzO. This result improves upon an earlier BadA study where the relative apparent maximum r ate ( ) was at 1% for Cyc compared with that for BzO, likely due to a low Cyc concentration (250 µM) ( 12 ) , below the calculated = 1470 µM in this study. 2.3.4 Rational Mutation of the BadA Active Site BadA structures in complex with carboxyla te substrates and a benzoyl - AMP intermediate guided site - directed mutagenesis on the wild - type BadA to remove steric bulk from the active site and expand the substrate preference for benzoates with meta - and para - substituents. Four residues (Ala227, Leu332 , His333, and Ile334) surround the phenyl ring of BzO in the active site ( Figure 2 . 5 ). The residue positioned near a given carbon of BzO depends on whether the BzO is in the carboxylate - bound orientation ( Figure 2 . 5 A), or the rotated Bz - AMP - bound orientation ( Figure 2 . 5 B). For example, Ala227 is next to the meta - carbon of BzO ( Figure 2 . 5 A) an d the ortho - carbon of Bz - AMP ( Figure 2 . 5 B). Similarly, the peptide backbone of Leu332 is near the para - and meta - carbons of BzO and Bz - AMP, 55 Table 2 . 2 . Relative app arent maximum rates of BadA and point mutants for various substrates . Substituent on BzO ( ) a BadA of BadA Point Mutations A227G I334A H333A L332A H (BzO) 100 b (8.8) c 86 (4) 93 (3) 89 (10) 89 (8) 2 - CH 3 10.6 (0.5) 42 (1) 1.8 (<0.1 ) 11 (1) 1.7 (0.2) 2 - NO 2 1.6 (0.10) 1.2 (0.012) 0.03 (0.002) 1.4 (<0.1) 0.12 (0.01) 2 - OCH 3 0.09 (0.008) 10.4 (0.33) 0.003 (<0.001) 0.058 (0.005) 0.004 (<0.001) 2 - CN 0.02 (0.002) 12.0 (0.82) 0.03 (<0.01) 0.08 (0.01) 0.05 (0.002) 2,6 - DiCl ND 0.69 (0.02) ND ND ND 3 - CH 3 2.04 (0.4) 7.0 (0.3) 49 (1.4) 44 (3.6) 1.5 (0.12) 3 - OCH 3 2.9 (0.32) 70 (6.2) 10.7 (0.24) 0.086 (0.009) 3 - CN ND 0.038 8.5 (1.3) 2.5 (0.15) 0.017 (0.002) 3 - NO 2 ND 0.015 (0.0023) 0.23 (0.036) 1.2 (0.034) ND 4 - CH 3 5.02 (0.304) 0.29 (0.014) 14 (0.35) 4.5 (0.202) 0.063 (0.004) 4 - CN 0.010 (0.001) 2.9 (0.3) ND 4 - OCH 3 ND < 0.001 39 (2.9) 0.93 (0.019) ND 4 - NO 2 ND ND 0.11 (0.010) ND 4 - Ethyl ND ND 9.4 (0.83) 0.86 (0.058) ND 3,4 - DiCH 3 <0.001 0.016 (0.0032) ND 0.69 (0.11) ND (PhAc) ND ND 0.012 (0.0022) < 0.001 ND a are normalized (as percentage (%)) to the apparent relative V max of BadA for benzoate. Highlighted (bold) are inactive substrates with BadA that are active with a mutant, or the mutant is 10 - f old more active than BadA. b for BadA with benzoate is 15.1 nmol/min. c Standard deviations are in parenthesis (n = 3). ND = Not Detected. 56 respectively. The His333 side chain points toward the meta - and para - positions of BzO and Bz - AMP, respect ively, while the Ile334 side chain is near the ortho - carbon of BzO and the meta - carbon of Bz - AMP. Therefore, targeted mutations of Ala227Gly, Leu332Ala, His333Ala, and Ile334Ala should show the relative importance of the two orientations for reactivity. Th e latter three were mutated to Ala instead of a smaller, flexible Gly to maintain conformational rigidity in the peptide backbone. Each mutant turned over the natural substrate benzoate at 86% to 93% the rate of BadA ( Table 2 . 2 ). 2.3.4.1 Ala227Gly - BadA mutant The maximum relative velocities ( ) of the Ala227Gly - BadA mutant increased for ortho - substituted BzO substrates (2 - Me (~4 - fold), 2 - CN (600 - fold), and 2 - MeO (116 - fold)) compared with those of BadA. Ala227Gly - B adA also uniquely converted 2,6 - dichlorobenzoate (2,6 - diCl - BzO) to its CoA thioester while BadA and the other mutant enzymes did not. Increases in activity with meta - substituted BzO substrates were also observed. A ~3 - fold increase in activity with 3 - Me - Bz O was observed in addition to a ~2900 - fold (or greater) increase in activity with 3 - MeO - BzO. Novel, but low level activities with 3 - CN - BzO and 3 - NO 2 - BzO were observed. Compared with the activities of BadA for substrates with para - substituents, the mutant a ctivities were roughly the same, except the mutation markedly decreased the turnover of 4 - Me - BzO by 17 - fold ( Table 2 . 2 ). 2.3.4.2 Ile334Ala - BadA mutant The Ile334Ala - BadA mutant had a higher for meta - and para - s ubstituted benzoates but lower for all ortho - substituted benzoates tested. The values of Ile334Ala - BadA compared with those of BadA increased 70,000 - fold for 3 - MeO - BzO, 24 - fold for 3 - Me - BzO, 2.8 - fold for 4 - Me - BzO, and 7 - fold for 4 - CN - BzO ( Table 2 . 2 ). The BadA Ile334Ala 57 mutant had expanded substrate specificity for previously inactive substrates: 4 - MeO (39%), 4 - ethylbenzoate (4 - Ethyl - BzO) (9.4%), 3 - CN - BzO (8.5%), 3 - NO 2 - BzO (0.2%), a benzoate homolog, phen ylacetate (PhAc) (0.01%) and 4 - NO 2 - BzO (0.001%) ( Table 2 . 2 ). The Ile334Ala mutation, designed to help binding and turnover of BadA for ortho - substituted substrates, dramatically increased the catalysis of larger me ta - and para - substituents 2.3.4.3 His333Ala - BadA mutant His333Ala - BadA mutant, designed to help binding and turnover of BzO substrates with substituents at the meta - position resulted in increases in activity with both meta - and para - substituted BzO substrates, but did not significantly change the for ortho - substituted BzO substrates compared to BadA. Compared with BadA, the values of His333Ala - BadA increased 11,000 - fold for 3 - MeO - BzO, 22 - fold for 3 - Me - BzO, 3,400 - fold for the 3,4 - diMe - BzO, an d 2,100 - fold for 4 - CN - BzO ( Table 2 . 2 ). New activities (relative to BzO (at 100%)) for this mutation include 3 - CN - (2.5%), 3 - NO 2 - (1.2%), 4 - MeO - (0.9%), 4 - NO 2 - (0.1%), and 4 - Ethyl - (0.9%) BzO substrates ( Table 2 . 2 ). 2.3.4.4 Leu332Ala - BadA mutant The Leu332Ala mutant was designed to increase binding and turnover of para - substituted benzoic acids. Upon testing this mutant, we observed a significant loss of activity (~90%) for nearly al l substrates except BzO. Small increases in activity with 3 - MeO - BzO and 3 - CN - BzO were observed. The Leu332Ala mutant turned over BzO at 89% the rate of BadA; other mutants tested turned over the natural substrate at a similar rate. 58 2.4 Discussion In this stud y, to provide insight on active site residues that define substrate selectivity, we solved the crystal structure of BadA and used this information to design point mutants with greater substrate permissivity. In addition, we measured the Michaelis parameter s of BadA for various substrates to evaluate how steric effects and electronics, through interactions with a heteroatom or substituent, affect binding and turnover. 2.4.1 BadA structure and homology Homologous adenylase structures of CoA ligases and AMP ligases (20 - 30% similarity) in the protein databank (including BadA) show that enzymes in this family typically fold into a larger N - terminal domain (400 - 550 residues) and a smaller C - terminal domain (~110 residues), where the active site lies between the domains. The N - terminal domain contains nearly all the residues that bind the carboxylate substrate. The N - terminal domain also binds the adenosyl group of ATP while the C - terminal domain residues coordinate the ribose and phosphate groups. Several related CoA lig ase crystal structures support a domain - alternation mechanism where the C - terminal domain rotates ~140° from its adenylation conformation to a thiolation conformation after the acyl - AMP forms and CoA binds ( 21 ) . The domain rotation occurs on a flexible hin ge loop that contains a conserved Asp residue ( 26 ) , and this conformational change configures the ligase for the thiolation step in the second half - reaction ( 44 ) . The adenylation conformation supposedly forms when the carboxylate alone (or with ATP) binds the active site in the N - terminal domain ( 24, 26 ) . After the formation of the acyl anhydride intermediate and binding of CoA, the C - terminal domain rotates into the thiolation conformation. This rotation places key 59 catalytic C - terminal residues in the acti ve site and primes the enzyme for thioesterification ( 21, 26, 45 ) . To our knowledge, all known structures of ATP - dependent aroyl CoA ligases in complex with only their natural carboxylate substrate adopt the adenylation conformation ( 14, 23, 25, 28 ) . The s tructure of a 4 - chlorobenzoate CoA ligase (CBAL) from Alcaligenes sp. AL3007 in complex with 4 - chlorobenzoate (4 - Cl - BzO) (PDB: 1T5D) or 4 - Cl - Bz - AMP (PDB: 3CW8) adopts the adenylation conformation ( Figure 2 . 6 A). Sub sequently, CBAL adopts the CoA - bound thiolation conformation when complexed with a phenacyl analog of 4 - chlorobenzoyl CoA and AMP (PDB: 3CW9) ( Figure 2 . 6 B). The crystal structure of a benzoate CoA ligase (BCL M ) (PD B: 2V7B) from Burkholderia xenoverans ( 14 ) (61% sequence identity with BadA), in complex with only benzoate, is in the adenylation conformation ( Figure 2 . 6 B). In contrast, all six BadA structures in complex with ei ther aryl carboxylates or Bz - AMP are found surprisingly in the thiolation conformation, even in the absence of CoA. This observation deviates from the predicted sequential domain - alternation mechanism proposed for ATP - dependent CoA ligases ( 21 ) . The conser ved Asp424 hinge residue in BadA and the flanking residues of the flexible loop are nearly identical with those in BCL M . Reasons for why the BadA and BCL M structures adopt different C - domain conformations when bound to aryl carboxylate substrates remain ob scure, particularly, since a BadA structure in the adenylation conformation is unavailable. However, one possible explanation is that the two enzymes have different resting states; BadA assumes the thiolation conformation, while BCL M adopts the adenylation conformation when benzoate is bound. 60 Further, sequence and structural alignments show that the active site residues (Figures A30 and A31) and architectures of the N - terminal domains of BadA and BCL M in complex with benzoate are identical. It is therefore interesting that the relative values for BadA with fluorobenzoates ranged between 75% and 117% relative to BzO (100%); whereas, BCL M turned over 2 - F - BzO at 2% the rate for BzO ( 14 ) . Active site residue Phe236 in the adenylation conformation of BCL M is located <4 Å above the carboxylate group of the bound BzO and blocks the CoA channel. The analogous Phe226 residue in BadA is offset by a ~72° rotation away from the BzO, opening the CoA - binding channel (Figure A .1. 31). Because the BadA and BCL M st ructures have different C - domain conformations, it is difficult to provide a structural rationale for differences in substrate specificity between BadA and BCL M . 61 A B C D Figure 2 . 6 . BadA s tructural alignments and C - terminal domain orientation. A. C - Terminal domains (ligands omitted for clarity) of CBAL in complex with 4 - Cl - BzO (PDB: 1T5D) (green) and 4 - Cl - Bz - AMP (PDB: 3CW8) (yellow), in the adenylation conformation, are overlaid with those of BadA in complex with BzO (PDB: 4EAT) (red) and Bz - AMP (PDB: 4ZJZ) (cyan) in the thiolation conformation. B. C - Terminal domains (ligands omitted for clarity) of CBAL in complex with both 4 - Chlorophenacyl CoA and AMP (PDB: 3CW9) (green), and BCL M in compl ex with BzO (PDB: 2V7B) (purple) are overlaid with that of BadA in complex with BzO (PDB: 4EAT) (red) in the thiolation conformation. C. Relative positions of catalytically important lysine residues in BadA and BCL M . C - terminal domain (blue) is in the thio lation conformation: Lys427 (green) of BadA (PDB: 4ZJZ) is in the active site and Lys512 (red) is solvent exposed. D. C - terminal domain (blue) is in the adenylation conformation: Lys520 (red) of BCL M (PDB: 2V7B) is in the active site and Lys433 (green) is solvent exposed. N - terminal domains (gray). 62 2.4.2 Catalytically important lysine residues in BadA Despite the different conformations of the C - terminal domain for BadA and BCL M , each uses a distinct C - terminal domain lysine (BadA: Lys427/512 and BCL M : Lys433/5 20) to orient the BzO substrate in either conformational state. In the adenylation conformation, BCL M uses Lys520 (aligns with Lys512 of BadA) to coordinate BzO. An earlier study showed that mutation to alanine or modification by native enzymatic N - acetylation of Lys512 of BadA reduced the benzoyl CoA product formation by 99% ( 30, 46 ) . In all BadA structures in the thiolation conformation presented here, Lys427 coordinates BzO in the active site and Lys512 is far from the active site and solvent - ex posed in the C - domain ( Figure 2 . 6 , C and D). The apo structure of the ACSM2A crystallized in the thiolation conformation, supporting the idea that this conformation is available for binding by the carboxylate ligan d ( 24 ) . A thiolation conformation resting state would make the conserved lysine (Lys512 of BadA), present in all ATP - dependent CoA ligases, solvent accessible for post - translational regulation by N - acetyltransferases/deacetylases ( 30, 46, 47 ) . The surface - exposed, conserved lysine residue is accessible to regulatory acetyltransferases/deacetylases when these ligases adopt the thiolation conformation. By contrast, the adenylation conformation return s this lysine to the active site for adenylation catalysis. Lys427 of BadA makes four polar contacts with Bz - AMP, one with the benzoyl oxygen, three with the AMP moiety, and two to Gly303 and Gly430 ( Figure 2 . 7 ). T hese contacts likely anchor Bz - AMP, and the positive charge of Lys427 primes the AMP - leaving group for CoA transesterification. Based on this information, we predicted a Lys427Ala - BadA mutant would slow the second thioesterification step of the BadA reacti on, and found indeed that the mutant was 99.9% less active compared to BadA. 63 However, the rate of acyl - AMP formation appears unaffected. In the absence of structural data, the importance of Lys427 of BadA on catalysis was unexpected due to the semi - conserv ed nature of the residue. Together, the earlier Lys512 modification study and the Lys427 mutagenesis described here suggest that when BadA assumes the adenylation conformation (likely stabilized when ATP binds), Lys512 enters the active site. It is imagine d that the latter residue then coordinates and primes BzO for nucleophilic attack on ATP based on homology with BCL M in the adenylation conformation. This mechanistic sequence is analogous to that described in an earlier study for ACSM2A ( 24 ) . The latter p roceeds through a proposed series of conformations [thiolation - (carboxylate - binding step), adenylation - (ATP - binding step), and returns to thiolation (acyl - AMP forming, CoA binding, and thioesterification steps)] to catalyze its thioesterification reactio n. Figure 2 . 7 . Active site of BadA showing possible polar contacts between Lys427 and Bz - AMP. T he Bz - AMP ligand (yellow), Gly304, and Gly431 (green) and d istances (Å) are shown. Heteroatoms are colored a s oxygen (red), phosphorus (orange), and nitrogen (blue). 64 Lys427 is not highly conserved among ligases, however BCL M contains a presumed functionally - similar lysine residue (Lys433) at the same position as the active site Lys427 of BadA, as does the phen ylalanine adenylation domain (PheA) of gramicidin synthetase I from Brevibacillus brevis , and the D - alanyl carrier protein ligase (DltA) from Bacillus cereus ( 28, 48 ) . In other ligases, such as the dihydroxybenzoate CoA ligase (DhbE) from Bacillus subtilis (Asn434) or 4 - chlorobenzoate CoA ligase (CBAL) (Ile406), other residues ( 23, 25 ) align with Lys427 of BadA (Figure A .1. 32). These residues are predicted to orient similarly to Lys427 of BadA in the thiolation conformation based upon sequence and structura l alignments. The importance of Lys427 for BadA catalysis of the thioesterification reaction led us to postulate that lysine surrogates of DhbE and CBAL perform a similar function in stabilizing the acyl - AMP intermediate. Likely, a combination of Asn434 an d Arg428 of DhbE and Asn411 and Arg400 of CBAL (similar to the highly - conserved Arg421 of BadA) form polar contacts with the acyl - AMP intermediates. We anticipate that lysine residues or nearby polar residues positioned similarly to that of Lys427 and Arg4 21 of BadA are important for thioesterification in other ATP - dependent CoA ligases. 2.4.3 Structural rationale for substrate specificity of BadA Through structural characterization of BadA, the different trajectories of BzO and the pathway intermediate Bz - AMP su ggest that the substrate must rotate ~60° counterclockwise (relative to re - face, see Figure 2 . 5 D) about the C 6 - axis of symmetry. This rotation causes the ring carbons of BzO to move past multiple active site residu es to access the Bz - AMP trajectory ( Figure 2 . 5 3.8 Å) to one ortho - carbon are Ile334 and Ala302. The other ortho - carbon points towards 65 a void within the active s ite where the phosphate group of Bz - AMP will ultimately reside ( Figure 2 . 5 and Figure A .1. 33), supporting the favored binding of ortho - substituted benzoates in BadA. The structures of ortho - substituted benzoates bo und to BadA all show them oriented such that the substituents occupy this void region, and are not pointed toward Ile334 and Ala302. The meta - carbons of the benzoates align with the Ala227 and His333, and the para - carbon is nearest the carbonyl of Leu332 o n the peptide backbone. The effects of the steric residues were evidenced generally by the higher values of BadA for meta - and para - substituents with increasing covalent van der Waals volumes F < OH < NH 2 < CN < Cl < Me < NO 2 < MeO of benzoates ( Table A2) over the ortho - regioisomers ( Table 2 . 1 ). When the substrate rotates toward the Bz - AMP conformation, the steric active site residues presumably reduced access to the catalytically proper orientation and ge nerally also slowed the of BadA for substrates with larger substituents ( Table 2 . 1 ). BadA typically turned over 2 - substituted substrates faster than the 3 - and 4 - substituted isomers. The observation is consistent with the kinetics data for substrates other than the F - BzO series. Fluorine is isosteric to hydrogen ( 43, 49 ) , and thus the steric demand of the F - BzO substrates is similar to BzO, as reflected in the similar values of BadA for 2 - and 4 - F - BzO and BzO. But overall, the enzyme turnover and binding affinity are principally affected by the regiochemistry and steric effects of the substituents ( Table 2 . 1 ). For instance, BadA only turned over 2 - MeO - Bz O presumably by placing the bulky methoxy group towards the open channel in the active site. We imagine the bulky methoxy group clashed with Ala227 before and after rotation of the benzoyl moiety of the substrate. As a 66 result, the binding affinity and turn over of BadA were reduced for 2 - MeO - BzO compared to BzO, and 3 - and 4 - MeO - BzO were not productive. Attempts to co - crystallize BadA with 4 - Me - BzO (98% purity) intriguingly resulted in a structure containing the 2 - methyl isomer (not shown). This isomeric sel ection strongly supported the idea that reduced steric interactions in the active site around one ortho - carbon of the substrate enabled highly regiospecific binding of ortho - substituted substrates. It is worth noting, that 2 - OH - BzO had the lowest of all substrates tested with BadA. Evaluation of the BadA - BzO complex suggests that the 2 - OH group may interact with the nearby hydroxyl of Thr329, creating a second binding contact ( Figure 2 . 5 D). In addition to interacting with the carboxylate of the bound BzO, Thr329 would provide an additional binding contact that may also affect release of the 2 - OH - Bz CoA as shown by the faster release of the 3 - and 4 - OH - Bz CoA products. Similarly, the regiochemistries of the splayed, planar NO 2 group and the extended, linear CN substituent likely prevented 3 - NO 2 - , 3 - CN - , the 4 - NO 2 - , and 4 - CN - BzO from binding and being turned over by BadA. As with the bulky 2 - MeO - BzO substrate, the 2 - NO 2 - and - CN - BzO were productive and convert ed slowly to their CoA thioesters by BadA ( Table 2 . 1 ). The linear 2 - CN substituent extends into the open channel of the active site ( Figure 2 . 5 , A and D) ( 43 ) . The for 2 - CN - BzO, however, is lower than nearly all of the 2 - substituted BzO substrates with smaller substituents, and this observation is consistent with the steric effects of Ala227 affecting catalysis as the substrate pivots during adenylation. To illus trate further, ortho - substituted substrates 2 - CN - BzO ( = 213 µM) and 2 - Cl - BzO ( = 130 µM) bind BadA similarly. However, the estimated covalent van der Waals length of cyano (C Ar - CN, d w = 3.1 Å) extends further than that of chloro (C Ar - 67 Cl, d w = 2.5 Å) (ChemBioDraw, ver. 13.0), and this difference may have reduced the of BadA for 2 - CN - BzO by 200 - fold compared with that for 2 - Cl - BzO. In summary, the size and position of the substituent on a BzO analog affect the relative rates of reactivity significantly more than the electronics of the substituent. BadA is more forgiving of ortho - substituents than para - substituents, consistent with the relatively open channel in the BadA structures next to the ortho - carbon of the BzO substrate. In contrast, substituents at the meta - and para - positions significantly impact activity, except for fluorinated substituents, which are isosteric with hydrogen and thus have no additional steric requirements. 2.4.3.1 Non - aromatic carbocycle carboxylates The Cyc subs trate bound BadA the least among the non - aromatic carbocycles likely because the staggered ring conformation and additional hydrogens clashed with active site residues. Estimated by , substrates 1 - Cyc and 3 - Cyc have ~12 to 13 - fold more apparent af finity for BadA than cyclohexane ( Table 2 . 1 ). The planarity of the double bond in these mono - unsaturated carbocycles likely reduced steric interfaces between the ring and active site residues within the BadA active site. The turnover of 1 - Cyc was lower than that for Cyc ( Table 2 . 1 ) while the placement of the double bond in 3 - Cyc strongly increased catalytic turnover. The double bond positioned between the meta - and para - carb ons of the ring in the latter removes two hydrogen atoms from the substrate that binds in a sterically crowded area near Ala227. Decreasing this steric hindrance likely explains the higher turnover and lower of 3 - Cyc relative to Cyc and 1 - Cyc ( Table 2 . 1 ). 68 2.4.4 Analysis of point mutants of BadA Mutational analyses also supported the substrate rotation mechanism in the BadA active site during catalysis. The four mutants Ala227Gly - BadA, Ile334Ala - BadA, His333Ala - B adA, and Leu332Ala - BadA in this study were designed to reduce the steric - clash encountered by the substituents during substrate binding and rotation to form Bz - AMP within BadA. For example, the proposed 60° rotation causes the ortho - and meta - carbons of th e initially bound BzO to move to the former locations of the meta - and para - carbons, respectively. Support for substrate rotation arises from a representative Ala227Gly - BadA mutation that reduced the steric interactions near the ortho - and meta - carbons of B zO. This mutant showed increased activity over BadA for ortho - substituted substrates that were imagined to bind and rotate within a sterically more favorable active site. This same mutant showed low but novel activities with meta - substituted BzO substrates , suggesting that the Ala227Gly mutation enabled meta - substituted substrates to now bind and rotate to a catalytically competent adenylation orientation ( Table 2 . 2 ). Collectively, the Ala227Gly - BadA, Ile334Ala - BadA , and His333Ala - BadA mutants turned over new substrates (3 - MeO, 3 - CN, 3 - NO 2 , 4 - MeO, 4 - NO 2 , 4 - Ethyl, 3,4 - DiCH3, and, surprisingly, phenylacetate (PhAc), a precursor of penicillin G) that BadA could not ( Table 2 . 2 ). Ile334Ala - BadA and His333Ala - BadA were active with nearly all of the 16 substrates tested, including PhAc, making these mutants broadly active 2 - , 3 - , or 4 - substituted benzoate CoA ligases. Ala227Gly - BadA uniquely turned over 2,6 - DiCl - BzO. The results of t he point mutations of BadA again supported a steric argument for BadA reactivity as opposed to one for substituent electronic effects. It is unclear why the Leu332Ala - BadA mutant, designed to reduce the steric interactions around the para - 69 carbon of the sub strate, unexpectedly did not turn over the para - substituted analog s, and slowed the turnover of several non - natural substrates tested ( Table 2 . 2 ). The peptide backbone carbonyl of Leu332 contributes to the active s ite architecture, while the Leu side chain is oriented away from the active site. The Leu side chain most likely engages in structural, hydrophobic interactions ( Figure 2 . 5 , A and B). Therefore, we suspect the Leu3 32Ala mutation may have malformed the active site of BadA. 70 2.5 C onclusion s Structures of BadA in complex with aryl carboxylate or aryl carbonyl - AMP show a persistent thiolation conformation before adenylation or CoA binding. This pre - CoA - bound conformation of BadA deviates from available structures of bacterial aryl carboxylate CoA ligases in the protein data bank. This suggests that the enzyme dynamics of BadA may be unique among benzoate CoA ligases during the substrate binding, adenylation, and CoA - thioes terification steps. This suggests two possible subclasses of benzoate CoA ligases, i ) those whose thiolation conformation is the resting state and therefore bind benzoate in this conformation, and ii ) those whose resting state is the adenylation conformati on, and bind benzoate in this conformation. Additional biochemical and structural analyses are required to further support this hypothesis. Further, similarity in the protein architecture and ligands bound, yet differences in the overall tertiary protein s tructure, aid in understanding the mechanisms of action. With this understanding, it was possible to design mutant constructs that were dramatically more permissive than the native protein. The Ala227Gly - BadA mutant improved catalysis primarily with ortho - substituted benzoates, while the Ile334Ala - BadA and His333Ala - BadA improved turned over with meta - and para - substituted benzoates over native BadA catalysis. These findings are important for making non - natural acyl CoA thioesters that can be used in a chas sis organism engineered to express natural product pathways that make next - generation bioactive compounds. Expanding the BadA substrate specificity may also help engineer pathways for bioremediation where substrate specificity is often a bottleneck for str ain development ( 50 ) against pollutants like polycyclic aromatic hydrocarbons ( 51 ) and polychlorinated biphenyls ( 52, 53 ) . In addition, the BadA kinetic constants provide data 71 needed for modeling flux control analysis in synthetic biology and bioengineerin g ( 54 - 57 ) , as well as metabolizing carboxylate - containing xenobiotic drugs in animals ( 24 ) . 72 APPENDIX 73 APPENDIX Figure A .1. 1 . Michaelis - Menten plot of bi osynthetic benzoyl CoA catalyzed by BadA (0.1 µ g/mL) from benzoate (BzO). V max = 0.25 ± 0.008 nmol/min, K M = 4.40 ± 0.65 µ M, k cat = 27.5 ± 0.87 s - 1 , ( n = 3). Figure A .1. 2 . Michaelis - Menten plot of biosynthetic thiophene - 2 - carbonyl CoA catalyzed by BadA (0.3 µ g/mL) from thiophene - 2 - carboxylate (Thio - 2 - C). V max = 0.13 ± 0.07 nmol/min, K M = 37 ± 5.7 µ M, k cat = 4.8 ± 0.23 s - 1 , ( n = 3). 74 Figure A .1. 3 . Michaelis - Menten plot of biosynthetic 3 - furoyl CoA catalyzed by BadA (0.1 µ g/mL) from 3 - Furoate (3 - Fur). V max = 0.058 ± 0.0043 nmol/min, K M = 70.8 ± 12.3 µ M, k cat = 6.74 ± 0.48 s - 1 , ( n = 3). Figure A .1. 4 . Michaelis - Menten plot of biosynthetic cyclohexanoyl CoA catalyzed by BadA (0.5 µ g/mL) from cyclohexane carboxylate (Cyc). V max = 0.62 ± 0.05 nm ol/min, K M = 1466 ± 213 µ M, k cat = 13.5 ± 1.07 s - 1 ( n = 3). 75 Figure A .1. 5 . Michaelis - Menten plot of biosynthetic 1 - cyclohexen - 1 - oyl CoA catalyzed by BadA (0.1 µ g/m L) from 1 - cyclohexen - 1 - carboxylate (1 - Cyc). V max = 0.051 ± 0.0022 nmol/min, K M = 52 ± 7.1 µ M, k cat = 5.6 ± 0.24 s - 1 . ( n = 3). Figure A .1. 6 . Michaelis - Menten plot of biosynthetic 3 - cyclohexen - 1 - oyl CoA catalyzed by BadA (0.4 µ g/mL) from 3 - cyclohexen - 1 - carboxylate (3 - Cyc). V max = 0.58 ± 0.04 nmol/min, K M = 56.2 ± 9.1 µ M, k cat = 16 ± 1.03 s - 1 ( n = 3 for [S] = 10, 20, 35, 200, otherwise n = 2). 76 Figure A .1. 7 . Michaelis - Menten plot of biosynthetic 2 - fluorobenzoyl CoA catalyzed by BadA (0.1 µ g/mL) from 2 - fluorobenzoate (2 - F - BzO). V max = 0.31 ± 0.0064 nmol/min, K M = 8.1 ± 0.91 µ M, k ca t = 34 ± 0.7 s - 1 , ( n = 3 for [S] = 5, 15, 50, 75, 100, 200, otherwise n = 2). Figure A .1. 8 . Michaelis - Menten plot of biosynthetic 3 - fluorobenzoyl CoA catalyzed by Ba dA (0.1 µ g/mL) from 3 - fluorobenzoate (3 - F - BzO). V max = 0.32 ± 0.013 nmol/min, K M = 73 ± 11 µ M, k cat = 35 ± 1.45 s - 1 , ( n = 3). 77 Figure A .1. 9 . Michaelis - Menten plot of biosynthetic 4 - fluorobenzoyl CoA catalyzed by BadA (0.1 µ g/mL) from 4 - fluorobenzoate (4 - F - BzO). V max = 0.2 ± 0.006 nmol/min, K M = 6.6 ± 0.9 µ M, k cat = 22 ± 0.66 s - 1 , ( n = 3 for [S] = 20, 100, 150, otherwise n = 2). Figure A .1. 10 . Michaelis - Menten plot of biosynthetic 2 - chlorobenzoyl CoA catalyzed by BadA (0.2 µ g/mL) from 2 - chlorobenzoate (2 - Cl - BzO). V max = 0.04 ± 0.001 nmol/min, K M = 126 ± 12.3 µ M, k cat = 2.21 ± 0 .068 s - 1 , ( n = 3). 78 Figure A .1. 11 . Michaelis - Menten plot of biosynthetic 3 - chlorobenzoyl CoA catalyzed by BadA (0.2 µ g/mL) from 3 - chlorobenzoate (3 - Cl - BzO). V max = 0.0061 ± 0.0002 nmol/min, K M = 289 ± 23 µ M, k cat = 0.33 ± 0.011 s - 1 , ( n = 3). Figure A .1. 12 . Michaelis - Menten plot of biosynthetic 4 - chlorobenzoyl CoA catalyzed by BadA (0.2 µ g/mL) from 4 - chlorobenzoate (4 - Cl - BzO). V max = 0.0054 ± 0.00022 nmol/min, K M = 83.2 ± 11.1 µ M, k cat = 0.29 ± 0.012 s - 1 , ( n = 3). 79 Figu re A .1. 13 . Michae lis - Menten plot of biosynthetic 2 - aminobenzoyl CoA catalyzed by BadA (1 µ g/mL) from 2 - aminobenzoate (2 - NH 2 - BzO). V max = 0.36 ± 0.01 nmol/min, K M = 21 ± 2.3 µ M, k cat = 3.9 ± 0.1 s - 1 , ( n = 3). Figure A .1. 14 . Michaelis - Menten plot of biosynthetic 3 - aminobenzoate CoA catalyzed by BadA (5 µ g/mL) from 3 - aminobenzoate (3 - NH 2 - BzO). V max = 0.82 ± 0.05 nmol/min, K M = 174 ± 32 µ M, k cat = 1.8 ± 0.1 s - 1 , ( n = 3). 80 Figure A .1. 15 . Michaelis - Menten plot of biosynthetic 4 - aminobenzoyl CoA catalyzed by BadA (5 µ g/mL) from 4 - aminobenzoate (4 - NH 2 - BzO). V max = 0.081 ± 0.00195 nmol/min, K M = 25.3 ± 3.01 µ M, k cat = 0.18 ± 0.0043 s - 1 , ( n = 3). Figure A .1. 16 . Michaelis - Menten plot of biosynthetic 2 - hydroxybenzoyl CoA catalyzed by BadA (0.1 µ g/mL) from 2 - hydroxyben zoate (2 - OH - BzO). V max = 0.058 ± 0.0009 nmol/min, K M = 1.6 ± 0.2 µ M, k cat = 0.633 ± 0.01, ( n = 3). 81 Figure A .1. 17 . Michaelis - Menten plot of biosynthetic 3 - hydroxybe nzoyl CoA catalyzed by BadA (5 µ g/mL) from 3 - hydroxybenzoate (3 - OH - BzO). V max = 0.81 ± 0.02 nmol/min, K M = 229 ± 18 µ M, k cat = 1.8 ± 0.04 s - 1 , ( n = 3). Figure A .1. 18 . Michaelis - Menten plot of biosynthetic 4 - hydroxybenzoyl CoA catalyzed by BadA (5 µ g/mL) from 4 - hydroxybenzoate (4 - OH - BzO). V max = 0.43 ± 0.02 nmol/min, K M = 160 ± 17 µ M, k cat = 0.9 ± 0.04 s - 1 , ( n = 3). 82 Figure A .1. 19 . Michaelis - Menten plot of biosynthetic 2 - methylbenzoyl CoA catalyzed by BadA (3 µ g/mL) from 2 - methylbenzoate (2 - Me - BzO). V max = 0.33 ± 0.012 nmol/min, K M = 44 ± 6 µ M, k cat = 1.2 ± 0.042 s - 1 , ( n = 3). Figure A .1. 20 . Michaelis - Menten plot of biosynthetic 3 - methylbenzoyl CoA catalyzed by BadA (3 µ g/mL) from 3 - methylbenzoate (3 - Me - BzO). V max = 0.15 ± 0.0050 nmol/min, K M = 215 ± 25.6 µ M, k cat = 0.54 ± 0.018 s - 1 , ( n = 3). 83 Figure A .1. 21 . Michaelis - Menten plot of biosynthetic 4 - methylbenzoate CoA catalyzed by BadA (5 µ g/mL) from 4 - m ethylbenzoate (4 - Me - BzO). V max = 0.32 ± 0.016 nmol/min, K M = 590 ± 73 µ M, k cat = 0.7 ± 0.035 s - 1 , ( n = 3). Figure A .1. 22 . Michaelis - Menten plot of biosynthetic 2 - cya nobenzoyl CoA catalyzed by BadA (5 µ g/mL) from 2 - cyanobenzoate (2 - CN - BzO). V max = 0.050 ± 0.0016 nmol/min, K M = 213 ± 22 µ M, k cat = 0.11 ± 0.0034 s - 1 , ( n = 3). 84 Figure A .1. 23 . Michaelis - Menten plot of biosynthetic 2 - methoxybenzoyl CoA catalyzed by BadA (20 µ g/mL) from 2 - methoxybenzoate (2 - MeO - BzO). V max = 0.021 ± 0.00096 nmol/min, K M = 789 ± 82 µ M, k cat = 0.011 ± 0.00052 s - 1 , ( n = 3). Figure A .1. 24 . Michaelis - Menten plot of biosynthetic 2 - nitrobenzoyl CoA catalyzed by BadA (20 µ g/mL) from 2 - nitrobenzoate (2 - NO 2 - BzO). V max = 0.5 ± 0.02 nmol/min, K M = 2015 ± 167 µ M, k cat = 0.27 ± 0.011 s - 1 , ( n = 3). 85 Figure A .1. 25 . Crystals of Rhodopseudomonas palustris benzoate CoA ligase (BadA in complex with BzO). Figure A .1. 26 . Overlay of BadA structures in complex wi th bound ligands. Substrate bound enzymes are as follows BzO (tan), 2 - F - BzO (purple), 2 - Me - BzO (salmon), 3 - Fur (green), Bz - AMP (blue), and Thio - 2 - C (pink) (Left panel). Active site magnified to show overlay of bound substrates (Right panel). 86 Figure A .1. 27 . Michaelis - Menten plot of benzoyl CoA catalyzed by the Lys427Ala - BadA mutant . Enzyme was (15 µ g/mL) with the benzoate (BzO) substrate . V max = 0.0011 ± 0.00002 5 nmol/min, K M = 1.5 ± 0.31 µ M, k cat = 0.05 ± 0.0011 s - 1 , ( n = 4). 87 A. B. C. D. E. Figure A.1. 28 . Benzoyl AMP (Bz - AMP) produced by BadA and the Lys427Ala - BadA mutant. A. Lys427Ala - BadA mutant and separately B. wild - type BadA with 1 mM benzoate (BzO) and 1 mM ATP at 31 °C for 45 min. Neg ative control assays C. incubated with 1 mM BzO, 1 mM ATP and no enzyme, D. BadA and 1 mM ATP, and E. Lys427Ala - BadA mutant and 1 mM ATP. Assays contained 250 µg/mL ligase, and the product mixtures were analyzed by reverse - phase HPLC with A 258 - monitoring o f the effluent. The Bz - AMP eluted at R t = 5.8 min. F. Tandem mass spectrometry (MS/MS, negative ion mode) fragmentation of the biosynthetic Bz - AMP ([M - H] = m / z 450) fraction isolated from the reverse - phase HPLC after incubation of the Lys427Ala - BadA mut ant with 1 mM BzO and 1 mM ATP (see panel A ). The MS/MS fragmentation was identical for the biosynthetic Bz - AMP fraction isolated from the reverse - phase HPLC after incubation of BadA with 1 mM BzO and 1 mM ATP (see panel B ). 88 Figure A.1.28 (cont d ) . F. Figure A .1. 29 . Shifted position of Tyr432 in BadA bound with bzAMP. The Tyr432 residue is shifted toward the phosphate oxygen (phosphorus: orange; phosphate oxygen: red) of the AMP anhydride a nd away from Arg421 to accommodate the Bz - AMP. 89 BCLM ...//...176 KAAATGCDDIAFWLYSSGSTGKPKGTVHTHANLYWTAELYAKPILGIAE -- NDVVFSAAK 233 BCLC ...//...177 KAVASGCDDIAFWLYSSGSTGKPKGTVHTHANLYWTAELYAKAILGIVE -- QDVVFSAAK 234 BadA ...//...166 KPAATQADDPAFWLYSSGSTGR PKGVVHTHANPYWTSELYGRNTLHLRE -- DDVCFSAAK 223 4CBL ...//...146 EDPQREPAQPAFIFYTSGTTGLPKAAIIPQR - AAESRVLFMSTQVGLRHGRHNVVLGLMP 204 BCLM LF FAY GLGNGLTFPLSVGATAILMAERPTADAIFARLVE ---- HRPTVFYGVPTLYANML 289 BCLC LF FAY GLGNGLTFPLSVGATAVLMAERPTPD AIFARLTR ---- HKPTVFYGVPTLYASML 290 BadA LF FAY GLGNALTFPMTVGATTLLMGERPTPDAVFKRWLGGVGGVKPTVFYGAPTGYAGML 283 4CBL LY HVV GFFAVLVAALALDGTYVVIEEFRPVDALQLVQQE ----- QVTSLFATPTHLDALA 259 BCLM VSPNLP -- ARADVAIRICTSAGEALPREIGERFTAHFGCE ILDG IGST EML HI FLSNRAG 347 BCLC ASPNVP -- AREDLALRICTSAGEALPREIGERFTARFGAEILDG IGST EML HI FLSNRAG 348 BadA AAPNLP -- SRDQVALRLASSAGEALPAEIGQRFQRHFGLDIVDG IGST EML HI FLSNLPD 341 4CBL AAAAHAGSSLKLDSLRHVTFAGATMPDAVLETVHQHLPGEKVNI YGTT EA M NS LYMRQPK 319 BCLM ...//...466 GLVKTRAFVVLKREFAPSEILAEELKAFVKDR - LAPHKYPRDIVFVDDLPKTATG K IQRF 524 BCLC ...//...467 GLVKTRAFVALKGEFVASDALADELKAFVKGR - LAPHKYPRDIVFVDDLPKTATG K IQRF 525 BadA ...//...461 GLTKPKAYVVPR --- PGQTLSETELKTFIKDR - LAPYKYPRSTVFVAELPKTATG K IQR F 516 4CBL ...//...440 WGQSVTACVVPR --- LGETLSADALDTFCRSSELADFKRPKRYFILDQLPKNALN K VLRR 496 BCLM KLREQ --- 529 BCLC KLREQL -- 531 BadA KLREGVLG 524 4CBL QLVQQVSS 504 Figure A .1. 30 . Par tial amino acid sequence alignment of selected benzoate CoA ligases . BCL M and BCL C from B. xenovorans and BadA from R. palustris (active site residues are highlighted in black), and a 4 - chlorobenzoate coenzyme A ligase (4 - CBAL, listed as 4CBL), from Alcali genes sp . Strain AL3007 (active site residues are highlighted in gray) are shown . The alignment was calculated with ClustalW2. Figure A .1. 31 . Comparison of the position of Phe226 of BadA (in the thiolation conformation) and that of Phe236 of BCL M (in the adenylation conformation) bound to benzoate. The phenylalanine residue rotates into the CoA binding site acting as a gate. In the adenylation conformation, the phenylalanine residue (BCL M ) blocks the CoA binding site. In the thiolation conformation, the phenylalanine residue (BadA) rotates out of the active site, opening the channel. 90 BadA YVRNDDGSYTYAGRTDDMLKVSGIYVSPFEIEATLVQHPGVLEAAVVGVADEHGLTKPKA 467 BCLm YCRLPNGCYVYAGRSDDMLKVSGQYVSPVEVEMVLVQHDAVLEAAVVGVD - HGGLVK TRA 472 bACS ARRDEDGYYWITGRVDDVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIPHAIKGQAIYA 561 yACS AAKDKDGYIWILGRVDDVVNVSGHRLSTAEIEAAIIEDPIVAECAVVGFNDDLTGQAVAA 570 4CBL AVWTPEGTVRILGRVDDMIISGGENIHPSEIERVLGTAPGVTEVVVIGLADQRWGQSVTA 446 DhbE VRLTRDGYIVVEGRAKDQINRGGEK VAAEEVENHLLAHPAVHDAAMVSMPDQFLGERSCV 474 PheA ARWLSDGNIEYLGRIDNQVKIRGHRVELEEVESILLKHMYISETAVSVHKDHQEQPYLCA 474 DltA - GYVENGLLFYNGRLDFQIKLHGYRMELEEIEHHLRACSYVEGAVIVPIKKGEKYDYLLA 443 :* ** . : * : *:* : : .: . Bad A YVVPRPG --- QTLSE ------- TELKTFIKDRLAPYKYPRSTVFVAELPKTATGKIQRF 517 BCLm FVVLKREFAPSEILA ------- EELKAFVKDRLAPHKYPRDIVFVDDLPKTATGKIQRFK 525 bACS YVTLNHGEEPSP ------- ELYAEVRNWVRKEIGPLATPDVLHWTDSLPKTRSGKIMRRI 614 yACS FVVLKNKSSWSTATDDELQDIKKHLVFTVRKDIGPFA APKLIILVDDLPKTRSGKIMRRI 630 4CBL CVVPRLGETLSADAL --------- DTFCRSSELADFKRPKRYFILDQLPKNALNKVLRRQ 497 DhbE FIIPRDEAPKAA ---------- ELKAFLRERGLAAYKIPDRVEFVESFPQTGVGKVSKKA 524 PheA YFVSEKHIPL ------------ EQLRQFSSEELPTYMIPSYFIQLDKMPLTSNGKIDRKQ 522 DltA VVVPG EHSFEKEF ------ KLTSAIKKELNERLPNYMIPRKFMYQSSIPMTPNGKVDRKK 497 . : * .:* . *: : Figure A .1. 32 . Partial amino acid sequence alignment of a broad selection of ATP - dependent adenylases and coenzyme A ligases. A portion of the C - terminal domain is shown to illustrate the complete conservation of Lys512 in BadA (magenta) and the partial conservation of Lys427 in BadA (cyan). BadA: benzoate coenzyme A ligase from Rhodopseudomonas palustris , BCL M : benzoate coenzyme A ligase from Burkholderia xenovorans LB400, bACS: Acetyl coenzyme A synthetase from Salmonella enterica , yACS: Acetyl coenzyme A synthetase from Saccharomyces cerevisieae , 4CBL: 4 - chlorobenzoate coenzyme A ligase from Alcaligenes sp. AL3007, DhbE: 2,3 - Dihydroxybenzoate adenylase from Bacillus subtilis , PheA: phenylalanine activating domain from gramicidin synthetase 1 from Brevibacillus brevis , DltA: D - alanyl carrier protein ligase from Bacillus cereus . Structures were aligned by Clustal Omega (EMBL - EBI). Figure A .1. 33 . Relative position of benzoate and benzoyl AMP in the BadA active site. Black dot on BzO (cyan) highlights the ortho - carbon position near the void in the BadA active site where the pho sphoryl group of Bz - AMP (yellow) resides. 91 Table A .1. 1 . X - ray Crystallography Data and Refinement Statistics. Substrate 2 - Methyl Benzoic acid 2 - Thiophene Carboxylic Acid 2 - Furoic Acid 2 - Fluoro - Benzoic acid Benzoic acid Benzoyl - AMP Space group P21 P21 P21 P21 P21 P21 a (Å) 58.659 58.644 58.637 58.885 58.650 58.615 b (Å) 94.808 94.609 95.110 95.749 96.02 95.611 c (Å) 95.346 95.701 95.330 98.605 95.38 95.706 (°) 90 90 90 90 90 90 (°) 104.92 104.92 104.88 110.43 104.65 104 .53 90 90 90 90 90 90 Molecules per Asymmetric Unit 2 2 2 2 2 2 Total reflection 442714 541417 344579 389465 705943 430613 Unique Reflection 115478 99162 93457 92681 93581 112317 Completeness (%) 96.9 (74.3) 97.5 (80) 98.54 (89.89) 97.82 (80.81) 98.6 (96.58) 96.34 (81.37) Average I/ 17.1 (1.35) 21.84 (1.97) 16.91 (1.61) 12.68 (1.55) 21.7 (2.73) 17.9 (2.0) R merge (%) 7.7 (44.3) 9.70 (47.2) 11.1 (57.0) 10.5 (47.5) 8.9 (63.7) 8.8 (43.4) Resolution (Å) (Last Shell) 50 - 1.63 (1.66 - 1.63) 50 - 1.72 (1.77 - 1. 72) 50 - 1.76 (1.79 - 1.76) 50 - 1.77 (1.81 - 1.77) 50 - 1.80 (1.84 - 1.80) 50 - 1.73 (1.73 - 1.70) R cryst / R free (%) 15.81/19.31 15.65 /19.37 18.58/22.93 15.91/20.17 15.5/19.1 15.37/19.2 RMSD From Ideal Values Bond Length ( Å) 0.0192 0.0181 0.022 0.0196 0.022 0.022 Bon d Angle (°) 1.9244 1.9736 1.966 1.961 1.969 1.981 Average B factor 21.264 25.67 21.754 19.3 19.69 18.812 Number of water molecules 577 603 632 738 655 647 PDB IDs 4RLQ 4RMN 4RM3 4RM2 4EAT 4ZJZ a Values in the parenthesis refer to the last resolution she ll. 92 Table A .1. 2 . Calculated van der Waals Volume ( V w ) Substituent V w (Å 3 ) F 8.0 HO 8.9 NH 2 11.8 CN 15.5 Cl 17.4 Me 19.9 NO 2 23.0 MeO 25.1 93 REFERENCES 94 REFERENCES 1. Schmelz, S., and Naismith, J. H. (20 09) Adenylate - forming enzymes, Curr. Opin. Struct. Biol. 19 , 666 - 671. 2. Conti, E., Franks, N. P., and Brick, P. (1996) Crystal structure of firefly luciferase throws light on a superfamily of adenylate - forming enzymes, Structure 4 , 287 - 298. 3. Schmelz, S. , Kadi, N., McMahon, S. A., Song, L., Oves - Costales, D., Oke, M., Liu, H., Johnson, K. A., Carter, L. G., Botting, C. H., White, M. F., Challis, G. L., and Naismith, J. H. (2009) AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosy nthesis, Nat. Chem. Biol. 5 , 174 - 182. 4. Storz, M. P., Brengel, C., Weidel, E., Hoffmann, M., Hollemeyer, K., Steinbach, A., Muller, R., Empting, M., and Hartmann, R. W. 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(2011) Cloning and characterization of a novel CoA - ligase gene from Penicilli um chrysogenum , Folia Microbiol. 56 , 246 - 252. 99 Chapter 3. Expression and purification of the Taxus cuspidata baccatin III: 3 - amino - 13 - O - phenylpropanoyl transferase (BAPT) 3.1 Introduction Biosynthesis of the anticancer drug paclitaxel requires multiple acyltransferases in the BAHD enzyme superfamily, named for the first four discovered enzymes: a benzylalcohol O - acetyltransferase ( B EAT), an anthocyanin O - hydroxycinnamoyl - transferase ( A HCT), an anthranilate N - hydroxycinnamoyl/benzoyltransferase ( H CBT), and deacetylvindol ine 4 - O - acetyltransferase ( D AT) ( 1 ) . BAHD enzymes catalyze aliphatic or aromatic acylation of N - or O - acyl acceptor molecules in plant and fungal secondary metabolism pathways ( Figure 3 . 2 ). These pathways include t he modification of phenolic compounds for cell wall biosynthesis ( 2 ) , anthocyanin ( 3 - 5 ) , volatile compounds ( 6, 7 ) , and phytoalexin ( 8 ) biosynthesis among others ( 1, 9, 10 ) . BAHD enzymes range in size from 48 to 55 kDa, have low sequence identity (~25 - 34 % ) between members, with diverse substrate specificities ( 1, 10 ) ( Figure 3 . 2 ). Despite vastly different substrate specificities, BAHD enzymes contain a number of conserved motifs including the catalytic HXXXD motif in the active site and the structural DFGWG motif in the C - terminal half of the enzyme ( Figure 3 . 3 ). Elucidation of the first BAHD crystal structure of vinorine synthase (and subsequent crystal structure) clarified the role of these highly conserved motifs ( 5, 11 - 14 ) . The conserved histidine of the HXXXD motif is proposed to act as a general base to deprotonate the acyl acceptor for nucleophilic attack on the acyl donor and the conserved aspartate orients away from the active site performing a structural role ( 11 ) ( Figure 3 . 1 ). 100 Figure 3 . 1 . Proposed mechanism of BAHD acyltransferases. I. The con served histidine in the HXXXD motif acts as a general base upon the acyl acceptor, which nucleophilically attacks the carbonyl of the acyl donor thioester. II. A tetrahedral intermediate is formed which subsequently collapses. III. The catalytic histidine is restored to a deprotonated state and the acyl acceptor is acylated. Coenzyme A (CoA - SH) is released. The conserved DFGWG motif is located in the C - terminal domain, away from the active site and appears to perform a structural role ( 11, 15 ) . The determi nation of additional crystal structures showed conservation of the global structure of these enzymes, the functional conservation of the motifs HXXXD and DFGWD, and the variability between active site residues to accommodate vastly different acyl donor and acceptor groups ( 5, 12 - 14 ) . For example, while vinorine synthase crystallized in the apo form, the sorghum ( Sorghum bicolor ) hydroxycinnamoyl transferase (HCT) crystallized in complex with the reaction products, p - coumaroyl shikimate and coenzyme A (CoA - S H) ( Figure 3 . 3 ) ( 13 ) . A large channel through the middle of the BAHD acyltransferase forms the active site. Both the acyl acceptor (shikimate) and the acyl donor ( p - coumaroyl CoA) enter the channel and face each ot her in close proximity to the conserved histidine residue (His162 in HCT) ( Figure 3 . 3 ). The peptide backbones of all five crystallized BAHD enzymes align well with each other. Although these acyltransferases may bi nd dramatically different substrates, they appear to utilize the same binding sites and the conserved catalytic histidine residue. 101 Figure 3 . 2 . Examples of BAHD enzyme pro ducts. The final acylation on each product is blue. 102 A. B. Figure 3 . 3 . BAHD structure of the Sorghum hydroxycinnamoyltransferase (H CT). A. HCT structure (blue) is shown with conserved HXXXD motif (cyan) and DFGWG motif (yellow). The active site channel is delineated by a black circle. B. HCT structure bound with the p - coumaroyl shikimate and CoA - SH (magenta) products of the acyltransf erase reaction. PDB: 4KEC 103 There are five BAHD acyltransferase steps in paclitaxel biosynthesis ( Figure 3 . 4 ). All five of these acyltransferases share 57 - 65 % sequence identity with each other ( Figure A.2.1 ). Seve ral have been purified and biochemically characterized including taxadiene - - ol - O - acetyltransferase (TAT) (16), taxane - - O - benzoyltransferase (TBT) (17, 18), 10 - deacetylbaccatin III 10 - O - acetyltransferase (DBAT) (19), baccatin III: 3 - amino - 13 - O - phenylpr opanoyl transferase (BAPT) ( 20 ) , N - debenzoylpaclitaxol - N - benzoyltransferase (NDTNBT) ( 21 - 23 ) . Figure 3 . 4 . Condensed paclitaxel biosynthesis from geranylgeranyl diphosphate s howing the BAHD acyltransferases in the pathway. TAT, taxadiene - - ol - O - acetyltransferase, TBT, taxane - - O - benzoyltransferase, DBAT, 10 - deacetylbaccatin III 10 - O - acetyltransferase, BAPT, baccatin III: 3 - amino - 13 - O - phenylpropanoyl CoA transferase, NDTNBT, N - debenzoylpaclitaxol - N - benzoyltransferase. Acyla tions - - shown in red. The larger arrow represents multiple reaction steps. 104 BAPT is more active with the pathway precursor, (3 R ) - - phenylalanyl CoA ((3 R) - - Phe CoA) acyl donor than the (2 R ,3 S ) - phenylisoserinyl CoA ((2 R ,3 S ) - Phe CoA) acyl donor. Characterization of BAPT activity with (2 R ,3 S ) - phenylisoserinyl CoA is important for developing a condensed paclitaxel biosynthesis that bypasses the need to identify , - Figure 3 . 4 ). The substrate specificity of BAPT, particularly with aryl - substituted phenylisoserines, has never been investigated. A number of active analogs of paclitaxel have been chemically synthesized with modifications at this position ( Table 3 . 1 ) . Investigations into BAPT substrate specificity would show the potential to produce paclitaxel and docetaxel analogs of interest with modified C13 - sidechains ( 20 ) . Previous studies with BAPT used crude lysate and estimated BAPT concentrations at 1% of total protein. Estimates of the K M for BAPT with baccatin III and (3 R ) - - phenylalanyl CoA ((3 R ) - - Phe CoA) were determined at 2.4 µM and 4.9 µM respectively ( 20 ) . The catalytic turnover of BAPT has never been determined. Previous research with BAHD acyltransferases purified protein by standard overexpression and chromatogr aphy techniques. Vinorine synthase was expressed as a polyhistidine - tagged enzyme and purified by nickel - nitriloacetic acid (Ni 2+ - NTA) affinity chromatography, anion - exchange chromatography, and gel - filtration chromatography ( 11, 24 ) . The HCT enzyme was al so expressed as a polyhistidine tagged protein and purified by nickel - affinity and cation - exchange chromatography at yields of 10.4 mg/L ( 13 ) . Other BAHD acyltransferases were also purified from bacteria, but in cases where BAHD expression in E. coli resul ted in insoluble protein, the yeast Pichia pastoris was successfully used to express the enzyme ( 4, 25 ) . In order to purify recombinant BAPT enzyme, both P. pastoris and E. coli were investigated for the recombinant expression of 105 soluble, active BAPT enzym e. This chapter describes the optimization of the expression and purification of BAPT. Expression and induction conditions are discussed, as well as various column chromatography purification methods. A coupled activity assay was also developed in order to follow BAPT activity during purification optimization due to the commercial unavailability of the substrate (3 R ) - - Phe CoA. 106 Table 3 . 1 . Biologically active analog s as active as paclitaxel with modifications in the C13 - phenylisoserinyl sidechain. R 1 R 2 R 3 Biological Activity Assay + Reference 2 - methyl - prop - 1 - enyl cyclohexyl propanoyl LCC6 1 , LCC6 - MDR 2 , MCF7 3 , NCI/ADR 4 ( 26 ) 2 - methylpropyl cyclohexyl propanoyl LCC6, LCC6 - M DR, MCF7, NCI/ADR 2 - methyl - prop - 1 - enyl t - butoxy propanoyl MCF7, NCI/ADR 2 - methyl - prop - 1 - enyl t - butoxy c - PrCO MCF7, NCI/ADR cyclohexyl t - butoxy H P388 5 , M 6 ( 27 ) trimethyl t - butoxy H B16 7 , M ( 28 ) 4 - chlorophenyl phenyl acetyl M, B16 ( 29 ) 4 - methoxyphe nyl phenyl acetyl M, B16 ( 30 ) 4 - fluorophenyl phenyl acetyl M, B16 cyclohexyl phenyl acetyl M, B16 ( 31 ) 2 - pyridyl phenyl acetyl M, B16 ( 32 ) 4 - pyridyl phenyl acetyl M, B16 2 - furyl phenyl acetyl M, B16 2 - methyl - prop - 1 - enyl phenyl acetyl J774.1 8 ( 33 ) 4 - fluorobenzyl t - butoxy acetyl J774.1 4 - fluorobenzoyl t - butoxy H J774.1 + Activities with the same or better activity than paclitaxel 1 LCC6 breast cancer cell line 2 LCC6 - MDR multidrug resistant breast cancer cell line (Pgp+) 3 MCF7 breast cancer cell li ne 4 NCI/ADR multidrug resistant human ovarian cancer cell line 5 P388 leukemia cell line 6 In vitro microtubule assembly assay 7 B16 melanoma cell line 8 J774.1 murine macrophage cell line. 107 3.2 Experimental 3.2.1 Materials Chloramphenicol (99%) and HEPES (>99%) we re obtained from Fluka/Sigma Aldrich (St. Louis, MO). MOPS (>99%) was obtained from Research Products International, Corp. (Mt Prospect, IL). ATP, DTT, IPTG, ampicillin, kanamycin, cobalt - affinity chromatography resin, and PMSF were purchased from Gold Bio (St. Louis, MO). The following reagents were purchased from New England Biolabs (Ipswich, MA); dNTPs, Phusion HF DNA polymerase, all restriction enzymes, and T4 DNA ligase. The QIAGEN Plasmid Mini Prep Kit and Gel Extraction Kit were obtained from QIAGEN (Valencia, CA). The following reagents were purchased from Promega (Madison, WI); PureYield Plasmid Mini Purification System and Wizard SV Gel and PCR Clean - Up System . Coenzyme A (95%) was obtained from Lee Biosolutions (St. Louis, MO). (3 R ) - - phenylalanine (98%) was purchased from Peptech (Burlington, MA). EDTA - free protease inhibitor cocktail tablets were purchased from Roche Life Sciences (Indianapolis, IN). All taxanes including baccatin III (>98%) and docetaxel (>98%) were purchased from Natland International Corporation (Research Triangle Park, NC). The PfuTurbo DNA Polymerase, zeocin, and E. coli strain BL21(DE3) were sourced from Invitrogen (Carlsbad, CA), The pET28a expression vector, Rosetta(DE3), and Rosetta(DE3)(pLysS) came from (No vagen, EMD Millipore, Billerica, MA ), 3.2.2 BAPT purification from Pichia pastoris 3.2.2.1 Cloning of pHisBAPT and pMycBAPT The bapt gene (Genbank: JF338879.1 ) was amplified with the following primers HisBAPT - - CAC GAA TTC AAA AAT GTC TAT GAA GAA GAC AGG TTC - 108 d HisBAPT - - CTT CTC GAG TCA ATG ATG ATG ATG ATG ATG TAA CTT TGA CGG ACA CAC TTT AG - and XhoI), a C - terminal 6X - histidine tag, and a stop codon onto the ends of the amplicon. Products were amplified by polymerase chain reaction (PCR) using PfU Turbo DNA conditions used an initial 95 °C for 4 min denaturation step, followed by 34 cycles at 95 °C for 1 min, 60 °C for 1 min, 72 °C for 4 min, and a final extension at 72 °C for 10 min. PCR products were analyzed by agarose gel electrophoresis (1% agarose in 1X TAE), double - digested with EcoRI and XhoI digested (New England Biolabs, Ipswich, MA), uctions, and gel purified using the Qiagen Gel Extraction Kit. The pPICZc plasmid (Invitrogen, Carlsbad, CA) was also double - digested and gel purified. The gel - purified EcoRI/XhoI cut PCR insert and pPICZc vector were ligated (3:1 insert: vector molar rati o) with T4 DNA ligase (16 hours at 14 °C) according to the E. coli (Invitrogen, Carlsbad, CA) and grown on LB plates (16 hour, 37 °C) containing 25 µg/mL zeocin. Positive clones were scre ened by PCR and analyzed by DNA sequencing (Michigan State University Research Technology Support Facility (RTSF) Genomics Core). The amino acid sequence of HisBAPT is listed in ( Figure A.2.2 ). The same cloning procedure was used to generate the pMycBAPT e xpression plasmid containing a C - terminal Myc epitope tag, followed by a polyhistidine tag. The pMycBAPT bapt insert was amplified with primers HisBAPT - Fwd (above) and MycBAPT - - CTT CCG CGG TAA CTT TGA CGG ACA CAC TT - tion sites. The MycBapt amplicon was ligated in EcoRI and SacII digested pPICZc ( Figure A.2.3 ). 109 3.2.2.2 BAPT expression in P . pastoris Expression vectors pHisBAPT and pMycBapt were linearized with the PmeI restriction enzyme and transformed into P. pastoris strai n X33 by electroporation according to the EasySelect Pichia (1% yeast extract, 2% peptone, 2% dextrose, 1M sorbitol) plates containing zeocin (100 and 1000 µg/mL) and grown at 30 °C. Co lonies were also screened for growth on glycerol or methanol as a carbon source and subsequently tested for BAPT expression according to methods included vortexing with glass beads (0.5 mm), use of a Beadbeater (BioSpec) with glass beads (0.5 mm), or sonication with a Misonix XL 2020 sonicator. Lysate was centrifuged at 18000g for 10 minutes. The supernatant was then centrifuged at 100000g for 1 hour. The final supernatan SDS - PAGE, Western blot, and activity assay. 3.2.2.3 BAPT Western blot Western blots were performed to detect BAPT protein containing an N - terminal or C - terminal poly - histidine tag. Samples were transferr ed from an SDS - PAGE gel onto a PVDF membrane (Immobilon) (12 hours at 4 °C, 30 V) gel in transfer buffer (25 mM Tris (pH 8.3), 192 mM glycine, 10% (v/v) MeOH). The PVDF membrane, was carefully removed, soaked in methanol, dried, rinsed with water, and incu bated with blocking solution (1% (w/v) BSA in 10 mM phosphate buffer (pH7.2), 0.9% (w/v) NaCl) for one hour, incubated with mouse anti - 6X His tag antibody conjugated to alkaline phosphatase (1:5000 dilution) (AbCam, Cat. No. ab81652, Cambridge, UK) in 1X T BS (50 mM Tris - Cl, (pH 7.6), 150 mM NaCl) buffer for 1 hour, rinsed with 1X TBS three times for 10 minutes each, then 110 color - developed by incubation with 1 - Step NBT/BCIP (ThermoFisher Scientific, Waltham, MA). Color development was stopped by rinsing with 1 X TBS, followed by a 10 min incubation with 50 mM EDTA in 1X TBS. 3.2.3 BAPT purification from Escherichia coli 3.2.3.1 Cloning of pNterBAPT The bapt gene was amplified (Pfu Turbo DNA polymerase) with the primers NterBAPT - - GTT CAT ATG AAG AAG ACA GGT TCG TTT GCA GAG TT - NterBAPT - - GAT GGA TCC TCA TCA TAA CTT TGA CGG ACA CAC TT - PCR conditions used an initial 95 °C for 4 min denaturation step, followed by 34 cycles at 95 °C for 1 min, 57 °C for 1 min, 72 °C for 4 min, and a final extension at 72 °C for 10 min. PCR products were analyzed by agarose gel electrophoresis (1 % agarose in 1X TAE), and gel purified using the QIAGEN Gel Extraction Kit. The pET28a plasmid and PCR amplified insert were double - digested with NdeI and HindIII, followed by agarose gel electrophoresis and gel purification (as described above). The gel - purified PCR insert and NdeI/HindIII cut pET28a vector were ligated and transformed as described above, into BL21(DE3) E. coli (Invitrogen, Carlsbad, CA) on LB plates containing kanamy cin (50 µg/mL). Positive clones were screened by PCR as described above. The amino acid sequence of expressed NterBAPT is listed in Figure A.2.4 . 3.2.3.2 Cloning of pCterBAPT Cloning of pCterBAPT was performed similarly to pNterBAPT, except for the following modif ications. The gene was amplified by PCR with two primer sets. Both PCR products were then combined, boiled, and re - annealed to make sticky - ended PCR products ( 34 ) . Primers were as follows: PCR1: CterBAPT - - AAG AAG ACA GGT TCG TTT 111 GCA GA - APT - - TTA ACT TTG ACG GAC ACA CTT TAG - and PCR2: CterBAPT - - CAT GAA GAA GAC AGG TTC GTT TGC - CterBAPT - - AGC TTT AAC TTT GAC GGA CAC ACT T - . PCR products were then combined to generate insert with compatible ends for cloning. PCR1 and 2 were combined and incubated at 95 °C for 5 minutes in a heat block, which was then removed and allowed to slowly cool to 40 °C. The PCR amplicon was then ligated into NcoI and HindIII digested pET28a. The amino acid sequence of expressed CterBAP T is listed in ( Figure A.2.5 ). 3.2.3.3 Cloning of pOptBapt The codon - optimized bapt gene ( Figure A.2.6 ) was ordered from Genscript (Piscataway, NJ), cloned in the pET28a expression vector for expression with a C - terminal polyhistidine tag. The amino acid sequence of expressed OptBAPT is listed in Figure A.2.6 . 3.2.3.4 Cloning of pMBP - CterBAPT and pMBP - NterBAPT To generate MBP - CterBAPT, the codon - optimized bapt gene (opt - bapt ) was amplified by PCR with two primer sets. Both PCR products were then combined, boiled, and re - an nealed to make sticky - ended PCR products as described above ( 34 ) . Primers were as follows: CterMBP - - GAT CCA TGG GCA AGA AAA CCG GT - - TCA GCC GGA TCT CAG TGG T - - - CAT GGG CAA GAA AAC CGG T - - AGC TTC AGC CGG ATC TCA GTG GT - - NterBAPT, the non - codon optimized bapt gene (Nter - bapt ) was made with the following primers: NterMBP - - GAT CCA TGG GCA GCA GCC ATC AT - - TCC TCA TAA CTT TGA CG G ACA C - - - CAT GGG CAG CAG CCA TCA T - 112 - AGG TTC CTC ATA ACT TTG ACG GAC AC - amplified with Phusion HF DNA polymerase (New England Biolabs) per the ons used an initial 95 °C for 4 min denaturation step, followed by 34 cycles at 95 °C for 1 min, 57 °C for 1 min, 72 °C for 1.5 min, and a final extension at 72 °C for 7 min. PCR products were then combined to generate insert with compatible ends for cloni ng as described above. Annealed sticky - ended bapt was gel - purified using the Wizard SV Gel and PCR Clean - Up System (Promega, Madison, WI). The pMAL - c2X plasmid (New England Biolabs, Ipswich, MA) was digested with BamHI and HindIII and gel - purified (as desc ribed above). The gel - purified PCR insert and BamHI/HindIII cut pMAL - c2X vector were ligated and transformed as described above. Transformants were selected on 100 µg/mL ampicillin. Positive clones were screened by PCR and DNA sequenced as described above. 3.2.3.5 E. coli strains The pNterBAPT plasmid was transformed into BL21(DE3) E. coli for initial purification experiments. pNterBAPT was also transformed into Rosetta (DE3) and Rosetta (pLysS) E. coli to express rare tRNA codons. Other expression plasmids, includ ing pCterBAPT, pOptBAPT, and pMBPCterBAPT were also transformed into these E. coli strains. 3.2.3.6 BAPT expression in E. coli BAPT was expressed from pNterBAPT, pCterBAPT, pOptBAPT, and pMBP - CterBAPT in E. coli . Growth temperatures were modified for optimal expre ssion during the initial growth phase, and also upon induction of protein expression by IPTG. A starter culture of E. coli supplemented with the appropriate antibiotic (50 µg/mL kanamycin or 100 µg/mL ampicillin, and 25 µg/mL chloramphenicol for Rosetta E. coli ) was grown overnight at 37 113 °C. Variations of growth and induction were tested. Variation 1: The starter culture was used to inoculate (7 mL) a 1 L culture, which was then grown at 37 °C (or 30 °C) to an OD 600 of 0.6, induced by IPTG addition, and fin ally the temperature was lowered to 18 °C for 18 hr. Variation 2. The starter culture was used to inoculate (7 mL) a 1 L culture which was then grown for 18 hours at 25 °C, followed by IPTG induction, and continued incubation for 1 to 2 hours. IPTG concent rations (50 µM to 1 mM) were varied in both growth variations and induction of BAPT was qualitatively observed by SDS - PAGE. 3.2.3.7 Nickel - affinity chromatography E. coli cell pellets were resuspended (3 mL/g (wet weight) ) in ice cold Lysis Buffer (50 mM NaPO 4 (pH 8), 300 mM NaCl, 15 mM imidazole, and 5% glycerol). EDTA - free protease resuspension was kept on ice, lysed in batches (15 g pellet = ~four liters culture), and sonica ted for 15 minutes in three cycles of 10 sec pulses followed by 20 sec rests with a Misonix XL 2020 sonicator. Lysed cells were centrifuged at 18,000 g for 20 minutes. The supernatant was further centrifuged in a Beckman Coulter Ultra Centrifuge at ~100000 g for 1.5 hours. The supernatant was loaded onto a Fast - Flow Ni 2+ NTA column (10 mL) (Qiagen) pre - equilibrated with Lysis Buffer at 4 °C. The column was washed with 5 column volumes (cv) of Lysis Buffer, and eluted with 3 cv of Lysis Buffer containing 250 m M imidazole. The eluent was concentrated in a Millipore Amicon Ultra 30 kDa cutoff concentrator and buffer exchanged with 30 mM MOPS, pH 8.0 with 5% glycerol (Buffer B). The procedure above was modified with Lysis Buffer containing 1 M NaCl or 10% glycerol . 114 3.2.3.8 Ammonium sulfate fractionation Lysate was prepared as described above with pNterBAPT in BL21(DE3) E. coli . Lysate was gently stirred on ice for 15 minutes. Ammonium sulfate was then slowly added in 5 - 10 % (w/v) increments. After each 10 % increment, the lysate was stirred for 20 minutes and centrifuged at 8000 g for 15 minutes. The supernatant was collected, placed on ice, and gently stirred. The next 10 % ammonium sulfate fraction was added and the process repeated. Fractions were collected at each step for analysis by SDS - PAGE and activity assays. 3.2.3.9 Ion exchange chromatography NterBAPT was partially purified by nickel - affinity chromatography, desalted, and concentrated prior to anion - exchange chromatography. Fast - Flow Q Sepharose (~25 mL) (GE Healthcare Bi o - Sciences, Pittsburgh, PA) was equilibrated in 30 mM MOPS, (pH 8), with 5% glycerol. Partially purified NterBAPT was added to the ion exchange resin, the flowthrough (FT = ~30 mL) and washes (Q1 - Q5 = 4 x 30 mL) were collected. Bound protein was eluted (Q E = ~45 mL) from the resin with 30 mM MOPS, (pH 8), with 5% glycerol and 1 M NaCl. Fractions were analyzed for activity and by SDS - PAGE for purity. Cation - exchange chromatography was performed identically to the anion - exchange chromatography described abov e. The resin was SP - sepharose (30 mL) (GE Healthcare Bio - Sciences, Pittsburgh, PA). Fractions were collected and analyzed for BAPT activity and by SDS - PAGE for purity. 115 3.2.4 Optimized NterBAPT expression and purification. Optimized purification of NterBAPT requi red three chromatography steps: nickel - affinity chromatography, anion - exchange chromatography, and cobalt - affinity chromatography. The nickel - affinity purification and anion - exchange purification were performed as described above using E. coli from 10 L (~ 45 g) of cell culture. However, the desalted Nter - BAPT eluent was concentrated (< 5 mL) prior to loading on the Fast Flow Q sepharose resin. Only the first two flowthrough fractions (Q1 and Q2) contained NterBAPT activity. These fractions were combined and directly loaded onto a cobalt - affinity column (1.5 mL) (Gold Bio, St. Louis, MO). Cobalt Wash Buffer was 30 mM MOPS, (pH 8), 300 mM NaCl, 15 mM imidazole, and 5% glycerol. Cobalt Elution Buffer was the same as the Cobalt Wash Buffer, but with 250 µM imidaz ole. The flowthrough (Co - FT), wash (5 cv) (Co - W), and elution (3 cv) (Co - E) were collected and analyzed for activity and purity by SDS - PAGE analysis. The final cobalt elution (Co - E) was desalted, concentrated in 30 mM MOPS, (pH 8), 5% glycerol, flash - froze n (40 µL aliquots) in liquid nitrogen and stored at - 80 °C. Quantification of the 53 kDa band corresponding to NterBAPT was determined by gel densitometry (see below). 3.2.5 MBP - BAPT expression and purification BAPT was expressed as a maltose binding protein (MB P) fusion protein in BL21(DE3) E. coli (Invitrogen, Carlsbad, CA) from both the pMBP - NterBAPT and pMBP - CterBAPT expression plasmids. A single colony was selected and used to inoculate a 100 - mL culture of LB media containing 100 µg/mL ampicillin. The cultur e was grown overnight at 37 °C. Aliquots of this starter culture (7 mL) were used to inoculate a flask of fresh LB media (1 L) containing 100 µg/mL ampicillin. MBP - BAPT was purified from 116 eight liters which were grown at 30 °C until OD 600 = 0.6. Gene expres sion was induced by 0.1 mM IPTG for ~16 hours at 18 °C. Bacteria were harvested by centrifugation at 8,000 g for 5 minutes. Pellets were resuspended in Buffer A (30 mM MOPS, (pH 8), 300 mM NaCl, 15 mM imidazole, 5% glycerol) at 3 mL/g pellet (wet weight). P MSF (100 mM stock in isopropanol) was added to the cell suspension at a final concentration of 1 mM. MBP - BAPT was purified by nickel - affinity chromatography as described above. The eluent was loaded onto amylose - binding resin (1 mL), pre - equilibrated with Buffer B. The amylose column was then washed with 5 cv of Buffer B and eluted with 3 cv of Buffer B containing 10 mM maltose. The eluted MBP - BAPT was further concentrated in a Millipore Amicon Ultra 30 kDa cutoff concentrator and buffer - exchanged with Buff er B to a volume of ~1 - 3 mL. MBP - BAPT was aliquoted (40 µL), flash - frozen in liquid nitrogen, and stored at 80 °C. Final concentrations were ~0.3 mg/mL (estimated by the Coomassie (Bradford) Protein Assays) (Thermo Scientific Pierce, Grand Island, NY). Pr otein was analyzed for purity by SDS - PAGE, band densitometry, and proteomics. 3.2.6 Band densitometry Purified protein was separated by SDS - PAGE (10% running gel) and stained by Coomassie blue or by silver. For band densitometry, purified BadA (see Chapter 2) ( 99%) was run in serial dilutions to generate a standard curve ( Figure A.2.8 ). Band densities were determined on a BioRad Molecular Imager Gel Doc XR+ using ImageLab software. Linear regression was used to generate a standard curve (Excel, Microsoft, Redmon d, WA). BAPT (band density at ~53 kDa) levels were calculated from this curve. Quantification of total protein 117 was determined by Coomassie (Bradford) Protein Assays (Thermo Scientific Pierce, Grand Island, NY). 3.2.7 Proteomics Purified BAPT samples were submit ted to the Michigan State University Research Technology Support Facility (RTSF) Proteomics Core for identification of co - purifying proteins with BAPT. 3.2.8 BAPT activity assays Coupled assays with PheAT, the (3 R ) - - Phe CoA ligase, were used to follow BAPT acti vity throughout purification. PheAT was prepared as previously described ( 35 ) . Assays were prepared on ice and incubated at 31 °C for 5 minutes prior to enzyme addition. The initial PheAT assay (200 µL) contained baccatin III (1 mM in acetonitrile, 10 % ( v/v)), followed by 200 mM MOPS (pH 8), 3.5 mM MgCl 2 , 1 mM ATP, 1 mM (3 R ) - - phenylalanine, 1 mM CoA, and PheAT (0.75 mg/mL). After one hour at 31 °C, the BAPT purification fraction (50 µL) was added and allowed to react for an additional two hours. The reaction was stopped by acidification (pH 5) with 8.8% formic acid, followe d by ethyl acetate (2 mL) addition. The docetaxel internal standard (1 µM) was added to the reaction, and extracted twice with EtOAc (2 × 2 mL). The organic layers were pooled and dried under a nitrogen stream. Coupled assay products were analyzed by LC - ES I - MS/MS with multiple reaction monitoring of the N - debenzoyl - - deoxypaclitaxel ion fragmentation [M + H] + = m / z m / z 509 and of the internal standard (docetaxel) fragmentation (1 µM) ion [M + H] + = m / z m / z 509. Product peaks were quantified by calculating the ratio of the N - debenzoyl - - deoxyp aclitaxel peak area to that of the docetaxel (1 µM) internal 118 standard. The amount of product formed was converted to units of activity (µmol/min). Total protein (mg) was quantified by Coomassie Bradford assays. 119 3.3 Results and discussion 3.3.1 BAPT expression in Pi chia pastoris Success with other BAHD family members led us to believe that P. pastoris may be a more suitable host for high - level, soluble expression of BAPT than E. coli ( 4, 25 ) . The bapt gene was cloned into the pPICZc expression vector as an N - terminal His - tagged (pHisBAPT) and a cMyc epitope - tagged (pMycBAPT) protein for methanol - inducible, cytoplasmic expression in the yeast, P. pastoris . The pPICZ vector was designed for stable integration in P. pastoris AOX1 (alcohol dehydrogenase) promo ter region in pPICZ AOX1 promoter to integrate the bapt gene such that expression is driven by the AOX1 promoter when grown with methanol as the sole carbon source. Multiple HisBAPT clones were screened for BAPT expr ession after 16 and 41 hours ( Figure 3 . 5 A). Minor increases in band density consistent with HisBAPT (~52 kDa) were observed in induced samples but not in background samples transformed with pPICZ empty vector ( Figure 3 . 5 B). Large - scale expression and the addition of nickel - affinity chromatography consistently showed no HisBAPT (~52 kDa) present by SDS - PAGE (data not shown). Unfortunately, expression of P. pastoris clones con taining integrated cMycBAPT was also not detectable, either by SDS - PAGE or western blot ( Figure 3 . 5 C and D). No protein bands consistent with cMycBAPT (54 kDa) were observed. BAPT activity assays were also negative . 120 A. B. C. D. Figure 3 . 5 . SDS - PAGE of whole cell lysate from expressions of BAPT clones from P. pastoris . Cells were induced for 16 hours or as labeled, with 0.5 % methanol a nd 1000 µg/mL zeocin. A. Expression of five clones of HisBAPT (52 kDa). B. Background expression with five clones containing pPICZ empty vector. C. Expression of five clones of MycBAPT (54 kDa). D. Representative western blot of MycBAPT clones with anti - 6X His tag antibody. Arrow indicates expected band location for expressed BAPT protein. The 6X - His tagged BadA, benzoate CoA ligase, was used as a positive control. Ladder is Kaleidoscope Prestained Protein Ladder (BioRad, Hercules, CA). 121 Despite successes with other BAHD family members, the P. pastoris expression system proved unsuccessful at producing active, soluble BAPT. Although multiple clones (~20 per construct) were tested for expression and activity we were never able to detect any BAPT by western b lot or activity assays with (3 R ) - - Phe CoA and baccatin III (data not shown). The persistent lack of activity in P. pastoris , combined with active BAPT expression in E. coli redirected purification efforts from yeast to bacteria. Figure 3 . 6 . NterBAPT expression in crude cell lysates. Four NterBAPT (52.7 kDa) clones in BL21(DE3) E. coli were screened for expression. Induction conditions included growth at 37 °C to an OD 600 of 0.6, addition of 0.25 mM ITPG, and growth at 18 °C for 16 hou rs. Uninduced (U) and induced (I) whole cell lysates were compared to empty vector, pET28a (1), NterBAPT Clone #5 (2), NterBAPT Clone #10 (3), Clone #15 (4), and Clone #20 (5). NterBAPT expression is observed strongly in Clone #20 and less so in Clone #5 a nd Clone #10. There is no expression in the empty vector control or in Clone #15. Positive control (+) is BadA, a benzoate CoA ligase. Ladder is PageRuler Prestained Ladder (Thermo Fisher, Waltham, MA). 122 3.3.2 BAPT expression and purification in E. coli 3.3.2.1 Express ion optimization in E. coli BAPT was expressed as an N - terminal polyhistidine - tagged protein from pNterBAPT, hereafter referred to as NterBAPT. Induced whole cell lysate showed a ~53 kDa band consistent with the expected size (52.7 kDa) of NterBAPT ( Figure 3 . 6 and Figure 3 . 9 ). Subsequent purification steps showed NterBAPT to be in inclusion bodies (data not Figure 3 . 7 . Sol uble expression of NterBAPT with varied IPTG concentrations. E. coli were induced with IPTG concentrations from 50 µM to 500 µM. Nickel - affinity column elutions are shown for each IPTG concentration. Samples were either induced for 1 hour or 18 hours at 18 °C. Arrows point to induced band at ~53 kDa. The band density decreases from 50 µM to 500 µM IPTG. shown). Although most of the enzyme appeared to be lost, some NterBAPT activity was retained in the clarified lysate. Efforts to improve the amount of solu ble protein included modifying growth and expression conditions, moving the polyhistidine tag from the N - to C - terminal (pCterBAPT), and the generation of an expression vector containing a C - terminal His - tagged codon - optimized bapt gene for expression in E . coli (pOptBAPT). Increasing induction time and varying IPTG concentrations did not substantially affect accumulation of soluble NterBAPT or CterBAPT ( Figure 3 . 7 ). Additionally, more soluble NterBAPT was found wit h low concentrations (50 µM) of IPTG ( Figure 3 . 7 ). There is no 123 apparent dif ference between expression induction for 3 hours or 18 hours with pOptBAPT, suggesting the low solubility of BAPT is not related to codon u sage in E. coli ( Figure 3 . 8 ). Based upon IPTG concentration and temperature induction studies, the optimal NterBAPT expression conditions were growth at 30 °C for 16 hours, and induction with 50 µM IPTG for 1.5 hou rs at 18 °C. Expression of BAPT in different E. coli cell types, including Rosetta(DE3) and Rosetta(DE3)(pLysS) ( 36 ) , which contain expression plasmids for tRNAs of rare codons in E. coli , were also not effective at increasing soluble BAPT (not shown). The se results are consistent with the expression of OptBAPT, where no change in soluble protein was observed, also suggesting that problems with BAPT solubility are not related to codon usage. A comparison of NterBAPT expression with a T. cuspidata N - debenzoy lpaclitaxel - Figure 3 . 8 . Expression and induction of OptBAPT in E. coli . Codon optimized, OptBAPT (52.1 kDa), expression levels in E. coli BL21(DE3) cells induced for 3 hours or 18 hours at 18 °C with 0.2 5 mM IPTG. Uninduced (U) and induced (I) whole cell lysates, the wash (W) fraction from nickel - affinity chromatography and elution fractions (1 - 6) were compared between 3 hour and 18 hour induced E. coli . Arrows point to the expected 52 kDa band correspon ding to OptBAPT expression. Ladder is PageRuler Prestained Ladder (Thermo Fisher, Waltham, MA) 124 N - benzoyltransferase (NDTNBT) ( 21 ) , a similar paclitaxel biosynthetic acyltransferase ( 10 ) , shows the differences in soluble expression between the two highly s imilar enzymes (56.5% identity and 71.4% similarity, ( Figure A.2.1 ), ( Figure 3 . 9 ). NDTNBT is more soluble than BAPT expressed from any of the constructs tested. The uninduced (U) and induced (I) whole cell lysates of NterBAPT and NDTNBT have similar expression patterns. Upon purification by nickel - affinity chromatography, it is apparent from examining the elution (E) fractions, that larger proportions of NDTNBT than BAPT is soluble and bound to the nickel - affinity r esin ( Figure 3 . 9 ). The low solubility of BAPT is likely due to its conformation in E. coli and interaction with cellular chaperones ( 37 ) . Figure 3 . 9 . Relative e xpression levels of NterBAPT and the Taxus N - benzoyltransferase (NDTNBT) by SDS - PAGE . Uninduced (U), induced (I) for 18 hours at 18 °C with 0.25 mM IPTG, whole cell lysates and the nickel - affinity column elution (E) fraction from each purification are show n. The arrow points to the induced ~53 kDa band of NterBAPT. NDTNBT is ~53 kDa. Both enzymes were prepared identically in the same pET28a plasmid background. Ladder is PageRuler Prestained Ladder (Thermo Fisher, Waltham, MA) 125 Figu re 3 . 10 . SDS - PAGE of NterBAPT purification by nickel - affinity chromatography. Fractions are as follows CL: clarified lysate, FT: flowthrough, W 1 : wash 1, W 2 : wash 2, E c : concentrated eluent. Bands consistent with NterBAPT (52.7 kDa) are visible near the labeled 55 kDa ladder band. The ladder is PageRuler Prestained Ladder (Thermo Fisher, Waltham, MA). 3.3.2.2 Purification by nickel - affinity chromatography NterBAPT was expressed in E. coli cells (either BL21(DE3), Ros etta, or Rosetta (pLysS)), and grown under optimal conditions as described above. The clarified lysate was applied to nickel - affinity resin where the flowthrough (FT), washes (W), and elution (E) fractions were analyzed by SDS - PAGE and for activity ( Figu re 3 . 10 and Figure 3 . 11 ). The eluent (E) was highly impure with numerous bands present on the gel ( Figu re 3 . 10 , Lane E c ). Most activ ity was retained on the column, with only 8% lost in the FT and W fractions ( Figure 3 . 11 A). However, 66% of activity was lost in the elution (E) fraction compared with the clarified lysate (CL). This loss of activi ty could not be accounted for with activity measured in the other fractions. The loss was reproducible and unaffected by increased glycerol concentration (5% to 10%) or increased NaCl concentration (0.3 M to 1 M). It is possible that imidazole affects the stability and decreases activity during elution with high (>200 mM) imidazole concentrations. Unfortunately, lower imidazole concentrations were insufficient to elute NterBAPT from the nickel - affinity resin ( Figure 3 . 11 B). 126 A. B. C. Fraction Protein in Assay (mg) Activity (µmol/min) Specific Activity (µmol/min mg) Specific Activity (Fold Change relative to CL) CL 0.59 2.77 × 10 - 5 4.69 × 10 - 5 1 FT 0.52 1.08 × 10 - 6 2.08 × 10 - 6 0.04 W 1 1.79 1.47 × 10 - 5 8.22 × 10 - 6 0.18 W 2 0.21 1.40 × 10 - 5 6.69 × 10 - 5 1.4 E c 0.24 3.68 × 10 - 4 1.53 × 10 - 3 33 Figure 3 . 11 . NterBAPT activity during nickel - affinity chromatography. Fractions are as follows CL: clarified ly sate, FT: flowthrough, W 1 : wash 1, W 2 : wash 2, E c : concentrated eluent. A. Bar graph showing the loss of total activity over the course of chromatography. B. Bar graph showing that NterBAPT binds nickel - affinity resin. Concentrations above 200 µM are requi red to elute all enzyme from the column. C. NterBAPT specific activity table of nickel - affinity purification fractions. 0 20 40 60 80 100 CL FT W1 W2 Ec % Total Activity Relative to CL Nickel - Affinity Chromatography Fractions 0 20 40 60 80 100 10 25 50 100 200 Normalized NterBAPT Activity (%) Nickel - Affinity Chromatography Imidazole Elutions (mM) 127 Figure 3 . 12 . Ammonium sulfate fractionation of NterBAPT clarified lysate. 3.3.2.3 Ammoni um sulfate precipitation Although nickel - affinity chromatography increased the specific activity of NterBAPT compared to the lysate (33 - fold), the elution was still impure, making it difficult to precisely quantify NterBAPT, an important consideration for determining Michaelis - Menten kinetic parameters ( Figure 3 . 11 C) . Ammonium sulfate precipitation was tested as a m eans of precipitating undesired contaminating proteins early in the purification. Ammonium sulfate was added to the lysate stepwise in 5% or 10% (w/v) increments and NterBAPT activity was measured ( Figure 3 . 12 ). NterBAPT activity decreased dramatically (~45%) after the addition of 20% (w/v) ammonium sulfate. Most b acterial proteins precipitated between 35% - 60 % (w/v) ammonium sulfate ( Figure 3 . 12 ) . Since BAPT activity disappears before precipitation of protein impurities, ammonium sulfate fractionation was abandoned as a me ans of increasing NterBAPT purity. 3.3.2.4 Ion exchange chromatography Because nickel - affinity chromatography was insufficient to purify NterBAPT, an additional chromatographic step was sought. The calculated pI for NterBAPT is 8.0, 0 20 40 60 80 100 10 20 25 30 35 40 Activity Remaining (%) Ammonium Sulfate (% (w/v)) 128 suggesting that the enzyme may bind to either anion or cation - exchange resin in solution. Both anion (Q Sepharose) and cation (SP Sepharose) exchange resins were tested as a second chromatography method with partially purified NterBAPT (nickel - affinity chromatography elution). Collecte d fractions were analyzed by SDS - PAGE ( Figure 3 . 13 ). NterBAPT appeared to bind the cation - exchange resin, but not the anion - exchange resin (data not shown). Protein impurities from the nickel - affinity purification step were retained on the anion - exchange resin. The flowthrough (Q) collected from anion - exchange chromatography contained BAPT activity while most impurities were retained on the column resin ( Figure 3 . 13 ). For th is reason anion - exchange chromatography was chosen as the second step in the NterBAPT protein purification. 3.3.3 Optimized NterBAPT p urification The NterBAPT purification was optimized with three chromatography steps: nickel - affinity, anion - exchange (Q resin), and cobalt - affinity. Purification by the nickel - affinity and anion - exchange chromatography was performed as described above. The addition of a final cobalt - affinity chromatography step substantially improved the NterBAPT purification ( Figure 3 . 13 ). Eluent from this column was 40 - 80% pure (on average) with several protein impurities observed on a silver - stained SDS - PAGE gel ( Figure 3 . 18 ). Analysis of the activity in each frac tion showed a definite increase in specific activity (~14000 - fold) despite the loss of some BAPT activity during nickel - affinity 129 A. B. C. Fraction Protein (mg) Activity (µmol/min) Spe cific Activity (µmol/min mg) Total Activity (% Relative to CL) Specific Activity (Fold Change relative to CL) CL 6444 4.20 × 10 - 3 6.52 × 10 - 7 100 1.0 FT 3536 1.98 × 10 - 4 5.61 × 10 - 8 4.7 0.1 Ni - E c 275 1.98 × 10 - 3 7.21 × 10 - 6 47 11.0 Q1 21.5 1.06 × 10 - 3 4.95 × 10 - 5 25 75.8 Q2 12.6 1.98 × 10 - 4 1.58 × 10 - 5 4.7 24.2 Q3 - 4 31.5 6.67 × 10 - 5 2.12 × 10 - 6 1.6 3.2 Q5 82.7 1.16 × 10 - 5 1.41 × 10 - 7 0.3 0.2 Q6 56.6 3.05 × 10 - 5 5.39 × 10 - 7 0.7 0.8 Co - E c 0.126 1.22 × 10 - 3 9.65 × 10 - 3 29 14800 Figure 3 . 13 . Purification of NterBAPT by multiple chromatography steps . A. The nickel - affinity column concentrated eluent (E c ), anion - exchange column flowthrough/wash (Q1 - 5), and elution (Q6) fractions are shown. B. Fractio ns Q1 and Q2 were pooled, concentrated (Q c ) and loaded onto cobalt - affinity resin. The flowthrough (FT), wash (W) and elution (Co - E) fractions are shown. The Co - E fraction contains a 53 kDa band consistent with NterBAPT. C. Specific activity of the NterBA PT purification. The activity in both Q1 and Q2 were combined prior to loading on a cobalt - affinity column. Fractions are labeled as follows: CL; clarified lysate, FT; flowthrough, W; wash, E c ; concentrated eluent, Q; flowthrough/wash fractions from the an ion - exchange column, Q c ; concentrated Q1 and Q2, Co - E; eluted protein from the cobalt - affinity column. Ladder (L) is the PageRuler Prestained Ladder (Thermo Fisher, Waltham, MA). 130 chromatography ( Figure 3 . 13 ). The dramatic increase in specific activity is a function of the low amounts of soluble BAPT and the large amount of E. coli processed during purification. From this three column purification, on average, 0.087 mg (1.65 nmol, MW = 52.7 kDa) of NterBAPT (account ing for ~40 - 80% purity based upon gel densitometry) was purified from 40 g (wet weight from ~ 8 L) of E. coli . Yields from this purification were approximately 0.0022 mg of NterBAPT per gram of E. coli (wet weight) ( Figure 3 . 13 ). The purified NterBAPT was concentrated and proteins were identified by proteomics with tandem mass spectrometry analysis ( Figure A.2.9 ). Results confirmed the presence of NterBAPT and identified the major impurities ( Table 3 . 2 ). The chaperones GroEL and DnaK were identified, which is not surprising considering the limited solubility of NterBAPT. Other impurities included a cAMP receptor protein, UDP - D - glucuronate dehydrogenase, and 23S rRNA m1g745 met hyltransferase ( Table 3 . 2 ). These proteins may have binding - affinity for NterBAPT, but why they co - purified remains unclear. Table 3 . 2 . Proteomics analysis of purifie d NterBAPT. NterBAPT Protein Impurity Size (kDa) GroEL 57 cAMP receptor protein 24 UDP - D - glucuronate dehydrogenase 74 DnaK 69 23S rRNA m1g745 methyltransferase 30 131 Figure 3 . 14 . Coupled assay schematic with PheAT and BAPT. PheAT produces (3 R ) - - phenylalanyl CoA (IV.) from (3 R ) - - phenylalanine (III.) which is a substrate for BAPT. Baccatin III (I.) acts as an acyl acceptor. BAPT transfers the (3 R ) - - phenylalanyl group to the 13 - O position on baccatin III to form the acylated product N - debenzoyl - - deoxypaclitaxel (II.). 3.3.4 BAPT activity assays A coupled enzyme assay was developed with a modified bacterial acyl CoA ligase to detect BAPT activity since the substrate (3 R ) - - Phe CoA is commercially unavailable. The TycA - phenylalanine epimerase/non - ribosomal peptide synthetase (NRPS), monomer from the tycrocidine synthetase A biosynthetic pathway in Bacillus brevis was recently discovered to have broader activity ( 35, 38, 39 ) . The wild - type PheATE was domain to prevent transfer of the amino acid to the growing tyrocidine peptide chain ( 35 ) . These modifications resulted in an a ctive enzyme, PheAT, capable of biosynthesizing 132 A. B. Figure 3 . 15 . Biosynthesis of N - debenzoyl - - deoxypaclitaxel by BAPT. A. Structure of N - debenzoyl - - deoxypaclitaxel ( m / z m / z 509). Loss of the (3 R ) - - phenylalanyl sidechain and an acetyl group yield a fragment of m / z 509. B. Representative LC - ESI/MSMS chromatogram showing the pr oduction of N - debenzoyl - - deoxypaclitaxel (3.0 minutes) ( m / z m / z 509 (black), docetaxel internal standard: m / z 808 m / z 509 (red)) by MBP - BAPT (Amylose column elution fraction) and PheAT coupled assays containing baccatin III, (3 R ) - - phenylalanine, ATP, and CoA substrates. Mass spectrometry performed on a Waters Quattro micro API LC/MS/MS (Waters, Milford, MA). (3 R ) - - Phe CoA from (3 R ) - - phenylalanine, ATP, and CoA, a required precursor for the BAPT biosynthetic reaction ( Figure 3 . 14 ). The coupled assay system produces N - debenzoyl - - deoxypaclitaxel in two steps. First, PheAT biosynthesizes (3 R ) - - Phe CoA. Second, BAPT catalyzes the transfer of the (3 R ) - - phenylalanyl moiety to baccatin III, a paclitaxel pre cursor ( Figure 3 . 14 ) ( 35, 40 ) . Assays were analyzed by LC - ESI - MS - MS chromatography to detect N - debenzoyl - - deoxypaclitaxel ([M + H] + = m / z 734) ( Figure 133 3 . 15 ). The major pr oduct, N - debenzoyl - - deoxypaclitaxel, elutes at ~3 minutes. However, a low - level, broad secondary peak elutes at ~2.4 minutes. This secondary peak was present in all assays containing (3 R ) - - Phe CoA and baccatin III, including PheAT coupled assays and ass ays with chemically synthesized (3 R ) - - Phe CoA, suggesting the broad peak is derived from assay products. Tandem mass spectrometry of each peak shows nearly identical fragmentation patterns which are consistent with N - debenzoyl - - deoxypaclitaxel ( 41, 42 ) ( Figure 3 . 16 ). It is possible that the broad 2.4 min peak ( Figure 3 . 16 B, C) represents isomers of N - debenzoyl - - deoxypaclitaxel ([M+H] + = m / z 734) ( 43, 44 ) . Isomerization is reported under neutral to basic conditions when paclitaxel epimerizes at the C7 position to form 7 - epi - paclitaxel and possibly at the C10 position as well ( 44 ) . BAPT activity assays are incubated at pH 8, followed by dilute acid quenching (pH 5) with 8. 8% formic acid in order to stop the reaction because of residual BAPT activity after ethyl acetate extraction. The assay products were exposed to both basic and acidic conditions in which some low - level degradation products may occur. The formation of mult iple low - level isomers of N - debenzoyl - - deoxypaclitaxel is consistent with the broad, poorly resolved peak at 2.4 minutes. Activity levels were quantified in each fraction by integrating the area under the major product peak at ~3 min. This assay was used to follow BAPT activity over the course of the purification. 134 A. B. C. D. E. Figure 3 . 16 . Representative LC - ESI/MS/MS of a BAPT assay. A. Structures and fragmentation of N - debenzoyl - - deoxypaclitaxel. Docetaxel sidechain mass is in parentheses. B. Total ion chromatogram for the fragmentation of N - debenzoyl - - deoxypaclitaxel ( m / z 734). C. Product ion spectrum of m / z 734 at 2.4 minutes. D. m / z 734 at 2.95 minutes and E. doc etaxel ( m / z 808) at 4.4 minutes. Total ion chromatogram for the docetaxel product ion spectrum is not shown. 135 3.3.5 MBP - BAPT expression and purification We intended to pursue kinetic analysis, paclitaxel biosynthetic assays, and crystallography with purified BAP T protein. Amounts of purified Nter - BAPT were too low for protein crystallization screens or for large - scale assays. Consequently, another method for soluble BAPT expression was sought. Fusion proteins such as the maltose binding protein (MBP) ( 45 ) , N util ization substance A (NusA) ( 46, 47 ) , thioredoxin (TRX) ( 48 ) , and SUMO ( 49 ) are commonly used for enhancing the solubility of proteins ( 50, 51 ) . We chose to investigate whether the addition of an MBP fusion partner would enhance the expression and solubilit y of BAPT compared with previous efforts. The bapt gene was cloned in the pMal - c2X expression vector with an N - or C - terminal 6X - His tag ( Figure 3 . 17 ). We hypothesized that the MBP - CterBAPT construct would be more efficient at binding nickel - affinity resin than MBP - NterBAPT due to accessibility of the polyhistidine tag. Initial expression tests confirmed higher soluble expression of MBP - CterBAPT. Therefore, the fusion protein was used for large - scale expression and purification optimization. Figure 3 . 17 . Domain structures of MBP - BAPT fusion proteins . MBP - NterBAPT and MBP - CterBAPT are shown above . Maltose binding protein (MBP), polyhi stidine tag (6X - His). 136 A. B. Figure 3 . 18 . Representative purification of MBP - BAPT . A. The enzyme was purified by nickel and amylo se affinity chromatography as labeled on an SDS - PAGE gel. Fractions are P 2 ; insoluble protein, CL; clarified lysate; FT; flowthrough, W 1 ; wash 1, W 2 ; wash 2, E; elution, E c ; concentrated elution. B. SDS - PAGE gel (silver - stained) showing purified NterBAPT ( 1) and MBP - CterBAPT (2) (52.7 and 95.2 kDa, respectively) from BL21(DE3) E. coli . Ladder is PageRuler Prestained Ladder (Thermo Fisher, Waltham, MA). The fusion protein MBP - CterBAPT was purified by nickel - affinity and amylose - affinity chromatography ( Figure 3 . 18 ). Active protein was detected in the lysate by activity assay, but inclusion bodies containing MBP - CterBAPT were still present in E. coli expressing the MBP - BAPT fusion protein ( Figure 3 . 18 A). Expression optimization included reducing the growth temperature from 37 °C to 30 °C for ~14 h to an OD 600 of ~2, followed by a 90 min IPTG (100 µM) induction. Nickel and amylose - affinity chromatography were performed in succes sion to yield 1.4 mg (14.7 nmol, MW = 95.2 kDa) of purified MBP - CterBAPT (accounting for ~50 % purity based upon densitometry) from 35 g (wet weight) of E. coli or ~0.039 mg/g (wet weight) ( Figure 3 . 18 ). This was a 10 - fold increase (mol BAPT per gram of E. coli ) in the amount of MBP - CterBAPT purified compared with the previous NterBAPT methodology. A proteomics analysis identified the major impurities as GroEL, DnaK, and 2 - oxoglutarate dehydrogenase E1 component ( Table 3 . 3 ). GroEL and DnaK are both 137 Table 3 . 3 . Proteomics analysis of purified MBP - BAPT. MBP - BAPT Protein Impurity Size (kDa) GroEL 57 DnaK 69 2 - oxoglutarate dehyd rogenase E1 component 105 chaperones that assist in protein folding ( 52 ) and 2 - oxoglutarate dehydrogenase E1 component is an enzyme in the primary metabolic tricarboxylic acid (TCA) cycle. Given the low solubility of BAPT, it is not surprising that chape rones co - purified with the enzyme even after passing through multiple columns. Reasons why these and other proteins were retained after two different affinity column purification steps remain unclear. These proteins likely have affinity for BAPT and possib ly the Ni 2+ - NTA or amylose resin. Yields of MBP - CterBAPT were an order of magnitude higher than NterBAPT with fewer purification steps. For these reasons, MBP - CterBAPT was used to determine the Michaelis - Menten kinetic constants with substrates of interes t including, (3 R ) - - Phe CoA, (2 R ,3 S ) - PhIS CoA, baccatin III and 10 - deacetylbaccatin III (Chapter 4). 138 3.4 Conclusion s Other BAHD acyltransferases were successfully expressed in both P. pastoris and E. coli at high yields for protein crystallography and kinetics experimen ts ( 5, 11 - 14 ) . BAPT expression was barely detectable in whole cell lysates of P. pastoris and acyltransferase activity was never detected. Definitive expression and activity of BAPT in E. coli whole cell lysates led us to abandon the P. pastoris expression system. Soluble BAPT levels were consistently low (~0.002 mg/g E. coli ), regardless of whether BAPT was codon - optimized or expressed as an N - or C - terminal polyhistidine - tagged protein. Ultimately, BAPT was purified as an N - terminal polyhistidine - tagged p rotein (NterBAPT) to >50 % purity (by SDS - PAGE band densitometry) requiring three sequential chromatographic steps, including nickel - affinity, anion - exchange, and cobalt - affinity chromatography. Proteomics analysis identified the major impurities as E. col i chaperones GroEL and DnaK. The low yields and limited solubility of NterBAPT were not compatible with the long - term goals of BAPT kinetic characterization for paclitaxel biosynthesis and protein crystallography. Maltose binding protein (MBP) is known t o increase the solubility of fused protein partners through an unidentified mechanism ( 45 ) . A C - terminal polyhistidine - tagged, MBP - BAPT fusion protein was hypothesized to increase the solubility of BAPT over previous methods. Fusion protein was expressed i n E. coli and purified by two sequential chromatographic steps, including nickel - affinity and amylose - affinity chromatography. MBP - BAPT was purified to >50 % purity (by SDS - PAGE band densitometry) at 1.4 mg (14.7 nmol, MW = 95.2 kDa) or ~0.039 mg/g (wet wei ght) with greater solubility and 10 - fold (mol BAPT per g of E. coli ) higher yields than any prior BAPT 139 expression system. Proteomics identified the major impurities as GroEL and DnaK, similar to the previous expression system. 140 3.5 Future Research Purified MBP - BAPT was used for kinetic characterization (Chapter 4) with the acyl donors, (3 R ) - Phe CoA and (2 R ,3 S ) - PhIS CoA, and the acyl acceptors, baccatin III and 10 - deacetylbaccatin III. MBP - BAPT was used to demonstrate the biosynthesis of N - debenzoylpaclitaxel (the penultimate paclitaxel precursor), N - debenzoyl - 10 - deacetylpaclitaxel (the penultimate docetaxel precursor) and N - debenzoyl - N - 2 - furanylpaclitaxel (a biologically relevant paclitaxel analog ) ( 53 ) . Future research with BAPT will include attempts at pro tein crystallization - alone and as an MBP fusion protein ( 54 - 56 ) . BAPT has unique characteristics that make a crystal structure of high interest. The conserved HXXXD motif in all BAHD members is naturally modified to GXXXD in the BAPT enzyme. How the subst rates bind the active site for catalysis without the conserved catalytic histidine residue is unknown. Structural information may help explain the significance of the natural GXXXD mutation and implications for BAPT catalysis. 141 A PPENDIX 142 APPEND IX NDTNBT MEKAGS - TDFHVKKFDPVMVAPSLPSPKATVQLSVVDSLTICRG - IFNTLLVFNAP - DN - BAPT MKKTGSFAEFHVNMIERVMVRPCLPSPKTILPLSAIDNMARA --- FSNVLLVYAANMDR - DBAT -- MAGS - TEFVVRSLERVMVAPSQPSPKAFLQLSTLDNLPGVRENIFNTLLVYNAS - DR - TAT --- M EK - TDLHVNLIEKVMVGPSPPLPKTTLQLSSIDNLPGVRGSIFNALLIYNAS - PSP TBT --- MGR --- FNVDMIERVIVAPCLQSPKNILHLSPIDN -- KTRG - LTNILSVYNAS - QRV : * :: *:* *. ** : ** :*. : * * :: * NDTNBT -- ISADPVKIIREALSKVLVYYFPLAGR LRSKEIGELEVECTGDGALFVEAMVEDTISVL BAPT -- VSADPAKVIREALSKVLVYYYPFAGRLRNKENGELEVECTGQGVLFLEAMADSDLSVL DBAT -- VSVDPAKVIRQALSKVLVYYSPFAGRLRKKENGDLEVECTGEGALFVEAMADTDLSVL TAT TMISADPAKPIREALAKILVYYPPFAGRLRETENGDLEVECTGEGAMFLEAMA DNELSVL TBT S - VSADPAKTIREALSKVLVYYPPFAGRLRNTENGDLEVECTGEGAVFVEAMADNDLSVL :*.**.* **:**:*:**** *:*****..* *:*******:*.:*:***.: :*** NDTNBT RDLDDLNPSFQQLVFWHPLDTAIEDLHLVIVQVTRFTCGGIAVGVTLP H SVC D GRGAAQF BAPT TDLDNYNPSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGANVYGSAC D AKGFGQF DBAT GDLDDYSPSLEQLLFCLPPDTDIEDIHPLVVQVTRFTCGGFVVGVSFC H GIC D GLGAGQF TAT GDFDDSNPSFQQLLFSLPLDTNFKDLSLLVVQVTRFTCGGFVVGVSFH H GVC D GRGAAQF TBT QDFNEYDPSFQQLVFNLREDVNIED LHLLTVQVTRFTCGGFVVGTRFH H SVS D GKGIGQL *::: .**::**:* *. ::*: : **********:.**. . . .*. * .*: NDTNBT VTALAEMARGEVKPSLEPIWNRELLNPEDPLH - LQLNQFDSICPPPMLEELGQASFVINV BAPT LQSMAEMARGEVKPSIEPIWNRELVKLEHCMP - FRMSHLQIIHAPVIEE KFVQTSLVINF DBAT LIAMGEMARGEIKPSSEPIWKRELLKPEDPLYRFQYYHFQLICPPSTFGKIVQGSLVITS TAT LKGLAEMARGEVKLSLEPIWNRELVKLDDPKY - LQFFHFEFLRAPSIVEKIVQTYFIIDF TBT LKGMGEMARGEFKPSLEPIWNREMVKPEDIMY - LQFDHFDFIHPPLNLEKSIQASMVISF : .:.******.* * ****:**::: : :. ::: : .* : * ::* NDTNBT DTIEYMKQCVMEECNEFCSSFEVVAALVWIARTKALQIPHTENVKLLFAMDLRKLFNPPL BAPT EIINHIRRRIMEERKESLSSFEIVAALVWLAKIKAFQIPHSENVKLLFAMDLRRSFNPPL DBAT ETINCIKQCLREESKEFCSAF EVVSALAWIARTRALQIPHSENVKLIFAMDMRKLFNPPL TAT ETINYIKQSVMEECKEFCSSFEVASAMTWIARTRAFQIPESEYVKILFGMDMRNSFNPPL TBT ERINYIKRCMMEECKEFFSAFEVVVALIWLARTKSFRIPPNEYVKIIFPIDMRNSFDSPL : *: :.. : ** :* *:**:. *: *:*. .::.** .* **: :* :*:*. *:.** NDTNBT PNGYYGNAIGTAYAMDNVQDLLNGSLLRAIMIIKKAKADLKDNYSRSRVVTNPYSLDVNK BAPT PHGYYGNAFGIACAMDNVHDLLSGSLLRTIMIIKKSKFSLHKEL - NSKTVMSSSVVDVNT DBAT SKGYYGNFVGTVCAMDNVKDLLSGSLLRVVRIIKKAKVSLNEHF - TSTIVTPRSGSDESI TAT PSGYYGNSIGTACAVDNVQDLLSGSLLRAIMIIKKSKVSLNDNF - KSRAVVKPSELDVNM TBT PKGYYGNAIGNACAMDNVKDLLNGSLLYALMLIKKSKFALNENF - KSRILTKPSTLDANM . ***** .* . *:***:***.**** .: :***:* *:. * : * . NDTNBT KSDNILALSDWRRLGFY EA DFGWG GPLNVSSLQR - LENGLPMFSTFLYLLPAKNKSDGIK BAPT KFEDVVSISDWRHSIYYEV DFGWG DAMNVSTMLQQQEHEKSLPTYFSFLQSTKNMPDGIK DBAT NYENIVGFGDRRRLGFDEV DFGWG HADNVSLVQHGLKDVSVVQSYFLFIRPPKNNPDGIK TAT NHENVVAFADWSRLGFDEV DFGWG NAVSVSPVQQ -- QSALAM QNYFLFLKPSKNKPDGIK TBT KHENVVGCGDWRNLGFYEA DFGWG NAVNVSPMQQQREHELAMQNYFLFLRSAKNMIDGIK : ::::. .*. . : *.***** . .** : . : : . * :: ..** **** NDTNBT LLLSCMPPTTLKSFKIVMEAMIEKYVSKV ----- BAPT MLMF - MPPSKLKKFK IEIEAMIKKYVTKVCPSKL DBAT ILSF - MPPSIVKSFKFEMETMTNKYVTKP ----- TAT ILMF - LPLSKMKSFKIEMEAMMKKYVAKV ----- TBT ILMF - MPASMVKPFKIEMEVTINKYVAKICNSKL :* :* : :* **: :*. :***:* Figure A.2.1 . Multiple sequence alignment of Taxus cuspidata acyltransferases. Sequence identity between enzymes is 57 - 65%. The conserved HXXXD and DFGWG motifs are underlined. BAPT is the only BAHD acyltransferase without the conserved histidine residue. Alignment was made with the MUS CLE alignment tool (EMBL EBI, Cambridge, UK). 14 3 1 MSMKKTGSFA EFHVNMIERV MVRPCLPSPK TILPLSAIDN MARAFSNVLL 50 51 VYAANMDRVS ADPAKVIREA LSKVLVYYYP FAGRLRNKEN GELEVECTGQ 100 101 GVLFLEAMAD SDLSVLTDLD NYNPSFQQLI FSLPQDTDIE DLHLLIVQVT 150 151 RFTC GGFVVG ANVYGSACDA KGFGQFLQSM AEMARGEVKP SIEPIWNREL 200 201 VKLEHCMPFR MSHLQIIHAP VIEEKFVQTS LVINFEIINH IRRRIMEERK 250 251 ESLSSFEIVA ALVWLAKIKA FQIPHSENVK LLFAMDLRRS FNPPLPHGYY 300 301 GNAFGIACAM DNVHDLLSGS LLRTIMIIKK SKFSLHKELN SKTVMSSSVV 350 3 51 DVNTKFEDVV SISDWRHSIY YEVDFGWGDA MNVSTMLQQQ EHEKSLPTYF 400 401 SFLQSTKNMP DGIKMLMFMP PSKLKKFKIE IEAMIKKYVT KVCPSKLPRK 450 451 HHHHHH 456 Figure A .2.2 . HisBAPT amino acid sequence for expression in P. pastoris . Size is 51.9 kDa. 1 MSMKKTGSFA EFHVNMIERV MVRPCLPSPK TILPLSAIDN MARAFSNVLL 50 51 VYAANMDRVS ADPAKVIREA LSKVLVYYYP FAGRLRNKEN GELEVECTGQ 100 101 GVLFLEAMAD SDLSVLTDLD NYNPSFQQLI FSLPQDTDIE DLHLLIVQVT 150 151 RFTCGGFVVG ANVYGSAC DA KGFGQFLQSM AEMARGEVKP SIEPIWNREL 200 201 VKLEHCMPFR MSHLQIIHAP VIEEKFVQTS LVINFEIINH IRRRIMEERK 250 251 ESLSSFEIVA ALVWLAKIKA FQIPHSENVK LLFAMDLRRS FNPPLPHGYY 300 301 GNAFGIACAM DNVHDLLSGS LLRTIMIIKK SKFSLHKELN SKTVMSSSVV 350 351 DVNTKFEDVV SISDWRHSIY YEVDFGWGDA MNVSTMLQQQ EHEKSLPTYF 400 401 SFLQSTKNMP DGIKMLMFMP PSKLKKFKIE IEAMIKKYVT KVCPSKLPRK 450 451 RPPAYV EQKL ISEEDL NSAV DHHHHHH 477 Figure A .2.3 . cMycHisBAPT amino acid sequence for expression in P. pastor is . The Myc epitope tag is underlined. Size of protein is 54.3 kDa. 1 MGSSHHHHHH SSGLVPRGSH MKKTGSFAEF HVNMIERVMV RPCLPSPKTI 50 51 LPLSAIDNMA RAFSNVLLVY AANMDRVSAD PAKVIREALS KVLVYYYPFA 100 101 GRLRNKENGE LEVECTGQGV LFLEAMADSD LSVLTDLDNY NPSFQ QLIFS 150 151 LPQDTDIEDL HLLIVQVTRF TCGGFVVGAN VYGSACDAKG FGQFLQSMAE 200 201 MARGEVKPSI EPIWNRELVK LEHCMPFRMS HLQIIHAPVI EEKFVQTSLV 250 251 INFEIINHIR RRIMEERKES LSSFEIVAAL VWLAKIKAFQ IPHSENVKLL 300 301 FAMDLRRSFN PPLPHGYYGN AFGIACAMDN VHDLLSGS LL RTIMIIKKSK 350 351 FSLHKELNSK TVMSSSVVDV NTKFEDVVSI SDWRHSIYYE VDFGWGDAMN 400 401 VSTMLQQQEH EKSLPTYFSF LQSTKNMPDG IKMLMFMPPS KLKKFKIEIE 450 451 AMIKKYVTKV CPSKL 465 Figure A .2.4 . NterBAPT amino acid sequ ence for expression in E. coli . Size is 52.7 kDa. 1 MKKTGSFAEF HVNMIERVMV RPCLPSPKTI LPLSAIDNMA RAFSNVLLVY 50 51 AANMDRVSAD PAKVIREALS KVLVYYYPFA GRLRNKENGE LEVECTGQGV 100 101 LFLEAMADSD LSVLTDLDNY NPSFQQLIFS LPQDTDIEDL HLLIVQVTRF 150 151 T CGGFVVGAN VYGSACDAKG FGQFLQSMAE MARGEVKPSI EPIWNRELVK 200 201 LEHCMPFRMS HLQIIHAPVI EEKFVQTSLV INFEIINHIR RRIMEERKES 250 251 LSSFEIVAAL VWLAKIKAFQ IPHSENVKLL FAMDLRRSFN PPLPHGYYGN 300 301 AFGIACAMDN VHDLLSGSLL RTIMIIKKSK FSLHKELNSK TVMSSSVVDV 350 351 NTKFEDVVSI SDWRHSIYYE VDFGWGDAMN VSTMLQQQEH EKSLPTYFSF 400 401 LQSTKNMPDG IKMLMFMPPS KLKKFKIEIE AMIKKYVTKV CPSKLKLAAA 450 451 LEHHHHHH 458 Figure A .2.5 . CterBAPT amino acid sequence for expressio n in E. coli . Size is 52.1 kDa. 144 atgggcaagaaaaccggtagctttgccgaatttcatgtcaatatgatcgaacgtgtgatg M G K K T G S F A E F H V N M I E R V M gtgcgtccgtgcctgccgtcgccgaaaacgattctgccgctgtcggcgattgataacatg V R P C L P S P K T I L P L S A I D N M gcgcgcgcctttagcaatgttctgctggtctacgcggccaatatggatcgcgtttccgca A R A F S N V L L V Y A A N M D R V S A gacccggctaaagtcatccgtgaagcactgtcaaaagtgctggtttattactatccgttt D P A K V I R E A L S K V L V Y Y Y P F gctggccgcctgcgtaacaaagaaaatggtgaactggaagtggaatgcaccggccagggt A G R L R N K E N G E L E V E C T G Q G gttctgttcctggaagcaatggctgattccgacctgtcagtgctgacggatctggacaac V L F L E A M A D S D L S V L T D L D N t acaatccgtcctttcagcaactgattttctcactgccgcaagataccgacattgaagat Y N P S F Q Q L I F S L P Q D T D I E D ctgcacctgctgatcgtccaggtgacccgctttacgtgcggcggtttcgtggttggcgcg L H L L I V Q V T R F T C G G F V V G A aatgtttatggtt ctgcgtgtgacgccaaaggctttggtcaattcctgcagtcgatggca N V Y G S A C D A K G F G Q F L Q S M A gaaatggctcgtggcgaagtgaaaccgagcattgaaccgatctggaaccgcgaactggtt E M A R G E V K P S I E P I W N R E L V aaactggaacattgcatgccgtttc gtatgtcccatctgcaaattatccacgccccggtg K L E H C M P F R M S H L Q I I H A P V attgaagaaaaatttgtccagaccagcctggtgatcaacttcgaaattatcaatcacatt I E E K F V Q T S L V I N F E I I N H I cgtcgccgtatcatggaagaacgtaaagaaagcctga gctcttttgaaatcgtggcagct R R R I M E E R K E S L S S F E I V A A ctggtttggctggcgaaaattaaagccttccagatcccgcattctgaaaacgtgaaactg L V W L A K I K A F Q I P H S E N V K L ctgtttgcaatggatctgcgccgtagtttcaacccgccgctgccgcatg gctactatggt L F A M D L R R S F N P P L P H G Y Y G aatgcctttggcattgcgtgtgcgatggataacgttcacgacctgctgtcgggtagcctg N A F G I A C A M D N V H D L L S G S L ctgcgcaccatcatgatcatcaaaaaatcgaaattcagcctgcataaagaactgaattca L R T I M I I K K S K F S L H K E L N S aaaaccgtcatgagttcctcagtcgtggatgtgaacacgaaatttgaagatgttgtctct K T V M S S S V V D V N T K F E D V V S attagtgactggcgtcatagtatctactatgaagttgatttcggctggggtgacgcgatg I S D W R H S I Y Y E V D F G W G D A M aatgtctctacgatgctgcagcaacaggaacacgaaaaaagtctgccgacctatttttct N V S T M L Q Q Q E H E K S L P T Y F S ttcctgcagagtacgaaaaacatgccggatggtattaaaatgctgatgtttatgccgccg F L Q S T K N M P D G I K M L M F M P P agcaaactgaaaaaattcaaaattgaaattgaagcgatgattaaaaaatatgtcacgaaa S K L K K F K I E I E A M I K K Y V T K gtctgtccgtctaaactgaagcttgcggccgcactcgagcaccaccaccaccaccactga V C P S K L K L A A A L E H H H H H H - Figure A .2.6 . Codon optimized bapt gene sequence . A C - terminal polyhistidine tag (optBAPT) (52.1 kDa) is attached and the translated protein sequence for expression in E. coli is shown (Genscript, Piscataway, NJ). 145 1 MKIEEG KLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ 50 51 VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDKL YPFTWDAVRY 100 101 NGKLIAYPIA VEALSLIYNK DLLPNPPKTW EEIPALDKEL KAKGKSALMF 150 151 NLQEPYFTWP LIAADGGYAF KYENGKYDIK DVGVDNAGAK AGLTFLVDLI 200 201 KNKHMNADTD YSIAEAAFNK GETAMTINGP WAWSNIDTSK VNYGVTVLPT 250 251 FKGQPSKPFV GVLSAGINAA SPNKELAKEF LENYLLTDEG LEAVNKDKPL 300 301 GAVALKSYEE ELAKDPRIAA TMENAQKGEI MPNIPQMSAF WYAVRTAVIN 350 351 AASGRQTVDE ALKDAQTNSS SNNNNNNNNN NLGIEGRISE FGSMGKKTGS 400 401 FAEFHVNMIE RVMVRPCLPS PKTILPLSAI DNMARAFSNV LLVYAANMDR 450 451 VSADPAKVIR EALSKVLVYY YPFAGRLRNK ENGELEVECT GQGVLFLEAM 500 501 ADSDLSVLTD LDNYNPSFQQ LIFSLPQDTD IEDLHLLIVQ VTRFTCGGFV 550 551 VGANVYGSAC DAKGFGQFLQ SMAEMARGEV KPSIEPIWNR ELVK LEHCMP 600 601 FRMSHLQIIH APVIEEKFVQ TSLVINFEII NHIRRRIMEE RKESLSSFEI 650 651 VAALVWLAKI KAFQIPHSEN VKLLFAMDLR RSFNPPLPHG YYGNAFGIAC 700 701 AMDNVHDLLS GSLLRTIMII KKSKFSLHKE LNSKTVMSSS VVDVNTKFED 750 751 VVSISDWRHS IYYEVDFGWG DAMNVSTMLQ QQEHEKS LPT YFSFLQSTKN 800 801 MPDGIKMLMF MPPSKLKKFK IEIEAMIKKY VTKVCPSKLK LAAALEHHHH 850 851 HH 852 Figure A .2.7 . MBP - CterBAPT (95.2 kDa) amino acid sequence for expression in E. coli . The MBP sequence is underlined. A. B. Figure A .2.8 . Quantification of BAPT by band densitometry. A. Representative band densities on a silver - stained SDS - PAGE gel. Lanes 1 - 7: Dilutions of the stan dard BadA (2.4 - 38 µg/mL), Lane A: NterBAPT (47 % purity). Lane B: NterBAPT (76% purity). Arrows represent visible protein bands. The NterBAPT band is labeled. B. Standard curve generated from BadA band densities. 0 10 20 30 40 0 0.01 0.02 0.03 0.04 Intensity ( 10 5 ) BadA (mg/mL) 146 Figure A .2.9 . SDS - PAGE gel of purified NterBAPT and MBP - CterBAPT for p roteomics analysis . NterBAPT (52.7 kDa) (lane 1) contains several proteins estimated at 70, 60, 53, and 25 kDa. MBP - CterBAPT (95.2 kDa) (lane 2) contains several proteins at 95, 70, 60, and 53 kDa. Ladder is the PageRuler Pr estained Ladder (Thermo Fisher, Waltham, MA). 147 REFERENCES 148 REFERENCES 1. St - Pierre, B., and De Luca, V. 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(2014) Crystal structure of the essential Mycobacterium tuberculosis phos phopantetheinyl transferase PptT, solved as a fusion protein with maltose binding protein, J. Struct. Biol. 188 , 274 - 278. 153 Chapter 4. Paclitaxel analog biosynthesis from baccatin III with a four - enzyme in vitro system and characterization of baccatin III - 3 - amino - 13 - O - phenylpropanoyl CoA transferase (BAPT) 4.1 Introduction Clinical approval of paclitaxel for refractory ovarian cancer in 1992 increased demand for this structurally complex drug. Its subsequent approval for additional cancer chemotherapy treatments includ ing multiple combination therapies, applications in restenosis treatments, and its growing usage in new markets further increased demand and production requirements ( 1 ) . As described in Chapter 1, paclitaxel purification from Taxus sp. yew trees is environ mentally unsustainable, and complete chemical synthesis is cost prohibitive ( 1, 2 ) . Current production of paclitaxel is largely supplied through suspended plant cell culture fermentation (PCF) and semi - synthetic methods (See Chapter 1). Although production by these techniques dramatically improved upon paclitaxel yields isolated from yew tree bark (0.001 0.05% w/w) shortages of paclitaxel and its analog s occur annually ( 3 ) . These shortage s negatively impact patient healthcare ( 2, 4 - 7 ) . Causes for these shortages are related to high demand, manufacturing quality problems, shortages of raw materials, stock - piling by large hospitals, and other economic disincentives ( 8 ) . 4.1.1 Engineering the pacl itaxel biosynthetic pathway Major efforts are underway to engineer the paclitaxel biosynthetic pathway in heterologous organisms including Escherichia coli , Saccharomyces cerevisiae , and Arabidopsis thaliana ( 9 - 13 ) . These efforts are limited by multiple fa ctors including incomplete characterization of the paclitaxel pathway ( Figure 1 . 4 ). For example, the biosynthesis of 154 10 - deacetylbaccatin III (10 - DAB) from taxadiene - - ol requires enzymes with unidentified substrat e specificities as well as an ambiguous order of the enzymatic reactions ( 14 - 19 ) . Additionally, genes for several pathway enzymes remain unidentified, - C20 - epoxidase, an oxomutase, a C9 - the Taxus baccata - - elicited Taxus cell cultures using amplified cDNA screening techniques ( 20 ) . Additional unidentified gene transcrip ts homologous to cytochrome P450 hydroxylases, epoxidases, oxidases, and oxomutases were found, but biochemical characterization and substrate specificity studies with these putative pathway enzymes remain daunting challenges ( 20 ) . Over 400 taxoids are der ived from the paclitaxel precursor taxa - 4(5),11(12) - diene to form a complicated secondary metabolite network ( 21 ) . Enzymes within this network are known to divert flux from paclitaxel biosynthesis ( 16, 22, 23 ) . Defining a linear biosynthetic route to pacli taxel with limited side - products is difficult given these concerns ( 19 ) . Engineering the paclitaxel pathway in bacteria or yeast has benefits over current plant cell fermentation methods. Many paclitaxel biosynthetic enzymes accept multiple substrates an - - products and increase flux to the desired product. Purification of the final product would likely be easier without hundreds of intermediates and by - products produced in Taxus cells. Characteristics required of such a system include an in - depth understanding of pathway enzymes in terms of activity, substrate specificity, 155 Figure 4 . 1 . Condensed scheme of paclitaxel biosynthesis. Abbreviations: IPPI, isopentenyl diphosphate - 5 - - ol - - hydroxylase, TAT, taxadiene - - ol - O - taxane - - hydroxylase, DBAT, 10 - deacetylbaccatin III 10 - O - acetyltransferase, BAPT, baccatin III: 3 - amino - 13 - O - - - phenylalanine CoA ligase, NDTNBT, N - debenzoyltaxol - N - axane - - - - - - - - hydroxylase, TBT, taxane - - O - benzoyltransferase, epoxidase*, oxomutase*, C9 - - hydroxylase*. Enzymes marked with (*) are unidentified. 156 cellular location and regulatory control ( 24 ) . Bacteria and yeast are genetically tractable, can be grown large - scale in a controlled environment, generally use an inexpensive carbon source, are scalable, and have simpler extraction and purification procedures than those from plant cells ( 24, 25 ) . A wealth of knowledge regarding metabolic engineering and synthetic biology already exists for bacteria and yeast. The most successful example of drug production via engineered S. cerevisiae is the antimalarial drug artemi sinin, originally from Artemisia annua ( 26 ) . Biosynthesis of the intermediate artemisinic acid was achieved at high yields (initially 100 mg/mL ( 27 ) , later 25 g/L ( 28 ) ) and then developed by semi - synthesis to artemisinin or artesunate, an analog ( 28 ) . This bioengineering approach to artemisinin synthesis is currently in production and proposed to reduce drug costs and make anti - malarial drugs more available worldwide ( 29 ) . To date, paclitaxel bio - engineering efforts have focused on early pathway steps ( Figure 1 . 4 ). Taxadiene synthase (TS) was first engineered in E. coli to successfully produce taxa - (4)5,(11)12 - diene (taxadiene) ( 30 ) . Heterologous expression of TS in plants and yeast was also successful ( 10, 11 ) . An a mbitious early effort to express eight - OH, TAT, TBT, and DBAT in S. cerevisiae demonstrated production of trace levels of taxadiene - - ol. Subsequent enzymatic products were undetectable and the fi rst C5 - oxidation step was concluded to be a bottleneck ( 10 ) . Later efforts produced taxadiene in E. coli at g/L ( 12 ) E. coli , the natural gene was fused to a Taxus reductase and mo dified in the transmembrane domains ( 12 ) . A 98% conversion of taxadiene to taxadiene - - ol (~50%) and a by - product, 5(12) - oxa - 3(11) - cyclotaxane (OCT) (~50%) were observed ( Figure 4 . 2 ). The OCT by - 157 Figure 4 . 2 . Products of an engineered chimeric cytochrome P450 taxadiene - - hydroxylase expressed in E. coli . The desired product, taxadiene - - ol, and a side product, 5(12) - oxa - 3(11) - cyclot axane (OCT), were produced in approximately equal amounts. tobacco ( 31 ) . However, no natural taxadiene - - ol was detected in tobacco plants. The by - product likely resul ( 12 ) . Whether the chimeric enzyme was better or worse than the wild - or the environment of a heterologous bacterial system is unclear. Challenges in engineering Taxus sp . cytochrome P450 monooxygenase enzymes to function in bacteria include removing the requirement for P450 hydroxylases to be membrane - bound on the endoplasmic reticulum in the plant cell, while retaining activity ( 32 - 35 ) . There are ten predicted oxidatio n steps required to biosynthesize paclitaxel. In order to successfully engineer the entire pathway in bacteria, all P450 hydroxylases, the epoxidase, and the oxidase would likely need to be engineered to optimize expression and activity. Partner reductases are generally also required for heterologous engineering with P450 hydroxylases ( 33 ) . Recently, a mutualistic co - culture of both E. coli and S. cerevisiae split early biosynthetic genes between the two microorganisms. E. coli produced and 158 secreted taxadie ne, the S. cerevisiae oxidized the taxadiene with two fusion P450 enzymes - - hydroxylase/reductase, the 10 - - hydroxylase - , and the acetyltransferase, TAT ( 13 ) . Isotopic labeling and mass spectrometry suggested the observed diol product originated from taxadiene ( 13 ) . If additional by - products are formed or substrate specifi city is altered during the enzyme engineering process, pathway flux and product purification will likely become increasingly complex and costly. Most current knowledge regarding the regulation of paclitaxel biosynthesis is the result of elicitor studies on Taxus cell cultures. Genetic transformation of Taxus sp. is developing, but not yet routine ( 36 - 38 ) . Methyl jasmonate is well - known to have positive effects on paclitaxel accumulation in cell cultures ( 39, 40 ) . Recent work used these effects to find sev eral transcription factor repressors common to seven paclitaxel biosynthetic genes ( 41 ) . 159 Figure 4 . 3 . Coupled enzyme biosynthesis of paclitaxel. I. (2 R ,3 S ) - phenylisose rinyl CoA production by PheAT. II. Sidechain attachment to baccatin III by BAPT to produce the docetaxel precursor (R 1 = H) (III). IV. BadA catalyzes the prod - N of N - debenzoylpaclitaxel to produce paclitaxel. Baccatin III: R 1 = acetyl group. 10 - Deacetylbaccatin III (10 - DAB): R 1 = H. 160 4.1.2 Proposed paclitaxel semi - biosynthesis One possible approach to biosynthesize paclitaxel uses readily available precursors, baccatin III and 10 - DAB, as starting materials in a 4 - step enzyme system to produce paclitaxel ( Figure 1 . 9 ). In lieu of finding and optimizing the unid - hydroxylase R ,3 S ) - phenylisoserine ((2R,3S) - PhIS) was discovered in PheAT (truncated TycA), a module of the non - ribosomal peptide synthase (NRPS), tyrocidine synthase ( 42 ) . B accatin III: 3 - a mi no - 13 - O - p henylpropanoyl CoA t ransferase (BAPT) attaches the sidechain to baccatin III and is active with (2 R ,3 S ) - phenylisoserinyl CoA ((2 R ,3 S ) - PhIS CoA), albeit at a lower rate than (3 R ) - - phenylalanyl CoA ((3 R ) - - Phe CoA) ( 43 ) . N - d ebenzoyl t axol - N - b enzoyl t ransferase (NDTNBT) performs the final N - benzoylation step with both N - debenzoyl - - deoxypaclitaxel and N - debenzoylpaclitaxel. A broad specificity benzoate CoA ligase (BadA) (see Chapter 2) was used to biosynthesize various acyl CoAs to serve as putative s ubstrates for NDTNBT. Engineering the genes encoding these four enzymes in a heterologous host will enable production of paclitaxel (and its analog s) ( Table 4 . 1 ). Paclitaxel analog s already approved and those in cl inical trials typically contain modified acyl groups. Precursors can potentially be biosynthesized using the four enzymes in this methodology ( Figure 4 . 4 ). The costs and complexity of engineering the entire paclita xel pathway are avoided, no new enzymes need to be characterized, and flux to side products is decreased. For example, BAPT is selective for PhIS - - - phenylalanyl CoA. It is unlikely that side - phenylalanine. 161 Figure 4 . 4 . Taxanes clinically approved and in clinical trials. A. The natural product paclitaxel. B. Docetaxel. C. Cabazitaxel. D. DJ - 927 (Tesetaxel) completed phase II clinical trials for several cancers. E. BMS - 27 5183 (Phase I terminated). F. TL - 00139 completed phase II clinical trials for malignant mesothelioma. G. MST - 997 (Phase I - terminated). H. TPI - 287 ongoing clinical trials for several cancers and (clinicaltrials.gov). 162 Table 4 . 1 . Simple analog s of paclitaxel with biological activity. R 1 R 2 Biological Activity Assay + Reference 3 - chlorophenyl Acetyl M. 1 , B16 2 ( 44 ) 4 - chlorophenyl Acetyl M, B16 4 - methylphenyl Acetyl M, B16 4 - methoxyphenyl Acetyl M, B16 2 - furyl* Acetyl M, B16 ( 45 ) 3 - furyl Acetyl M, B16 2 - thiophen Acetyl A2780 3 , PC3 4 ( 46 ) 4 - methylphenyl H B16, P388 5 ( 47 ) 2 - fluorophenyl H B16, P388 4 - f luorophenyl H B16, P388 4 - chlorophenyl H B16, P388 4 - methoxyphenyl H B16, P388 + Activities with the same or better activity than paclitaxel * Analog biosynthesized in this work 1 In vitro microtubule assembly assay 2 B16 melanoma cell line 3 A2780 ovari an cancer cell line 4 PC3 prostate cancer cell line 5 P388 leukemia cell line 4.1.3 Applications in paclitaxel analog production Substrate specificity studies show PheAT is active with a number of arylisoserines, including meta - substituted (2 R ,3 S ) - phenylisoserin es, precursors for paclitaxel analog s with demonstrated cytotoxicity against multiple drug - resistant (MDR) cancer cell lines or currently in clinical trials ( 45, 48 - 54 ) . Substrate specificity studies with a benzoate CoA ligase (BadA), (see Chapter 2) and N - benzoyltransferase (NDTNBT) have also been performed ( 55, 56 ) . BadA has broad substrate specificity and single point mutations showed novel substrate activities as well. The Taxus N - benzoyltransferase (NDTNBT) is active with the following acyl acceptors: N - debenzoyl - - deoxypaclitaxel, N - debenzoyl - - deoxy - 10 - deacetylpaclitaxel, and N - debenzoylpaclitaxel. The acyl donor activity of 163 NDTNBT includes ortho - , meta - , and para - substituted benzoic acids with a preference for meta - or para - substituted benzoic acid s ( 55 ) . In addition to producing paclitaxel, it is likely this pathway can be used to produce analog s, or precursors to analog s of biological interest by simply changing the growth media to include relevant precursors such as baccatin III or 10 - DAB, phenyl isoserines, and benzoate surrogates. Many synthetic analog s have been tested in vitro but not further developed. 4.1.4 BAPT characterization and proof - of - principle for paclitaxel/analog biosynthesis One aim of this work was to biochemically characterize BAPT wi th (2 R ,3 S ) - PhIS CoA. Although BAPT is more active with (3 R ) - - Phe CoA, paclitaxel is biologically inactive - hydroxyl group ( 57 ) . In order to characterize Michaelis - Menten kinetic constants of BAPT, various synthetic and biosynthetic strategie s were employed to obtain (2 R ,3 S ) - PhIS CoA in preparative yields for the first time. BAPT kinetics were determined with both (3 R ) - - Phe CoA and (2 R ,3 S ) - PhIS CoA. Proof - of - principle for the biosynthesis of N - debenzoylpaclitaxel and paclitaxel analog s are de monstrated. 164 4.2 Experimental 4.2.1 Materials Coenzyme A (95%) was obtained from Lee Biosolutions (St. Louis, MO). (3 R ) - - phenylalanine (98%) was purchased from Peptech (Burlington, MA). (2 R ,3 S ) - phenylisoserine (98%) was purchased from Waterstonetech, LLC (Carmel, IN). All taxanes (baccatin III (>98%), 10 - deacetyl baccatin III (>98%), docetaxel (>98%) and paclitaxel (>98%) were purchased from Natland International Corporation (Research Triangle Park, NC). Additional reagents were sourced as follows: HEPES (>99%) (Fl uka/Sigma Aldrich, St. Louis, MO), MOPS (>99%) (Research Products International, Corp., Mt Prospect, IL), TEA (100%) (J. T. Baker, Center Valley, PA), acetic anhydride (>99.4%) and trifluoroacetic acid (>99.5%) (EMD Chemicals, Billerica, MA), di - t - butyl - di carbonate (>99%) and ethyl chloroformate (97%) (Sigma Aldrich, St. Louis, MO), C18 silica gel resin (carbon 23%, 40 - 63 µm) (Silicycle, Quebec City, Quebec, Canada). ATP, - D - 1 - thiogalactopyranoside (IPTG), kanamycin, phenylmethylsulfonyl fluoride (PMSF), and tris(2 - carboxylethyl)phosphine HCl (TCEP) were purchased from Gold Bio (St. Louis, MO). 4.2.2 Chemical synthesis of (3 R ) - - phenylalanyl CoA Synthesis of (3 R ) - - phenylalanyl CoA was adapted from a mixed anhydride intermediate method ( 43, 58 - 60 ) . The amine group of (3 R ) - - phenylalanine was protected as follows; (3 R ) - - phenylalanine (~243 µmol) was dissolved in 3:2 (v/v) 0.5 M NaHCO 3 : t - butanol (5 mL) and stirred at room temperature for 20 min. The solution was stirred on ice for 10 min prior to the dro pwise addition of di - t - butyl dicarbonate (2 equiv). After 30 min, the flask was removed from ice and stirred at 22 °C for 2 to 4 h and followed by TLC (8:1:1 butanol: 165 water: acetic acid). Upon completion, water (5 mL) was added to the flask and adjusted to pH 2 with 1M HCl. The reaction was then extracted three times with ethyl acetate (20 mL), the organic fraction was dried (anhydrous Na 2 SO 4 ), filtered, and evaporated under vacuum to yield R - N - Boc - 3 - amino - 3 - phenylpropanoic acid as a white powder (40.1 mg, 99% yield). 1 H - NMR (500 MHz, CDCl 3 ) (ppm): 1.43 (m, H5), 2.88 (m, H1, H2), 5.12 (br d, H3), 7.30 7.36 (aromatic protons). ( Figure A.3.1 for proton numbering). 3 R - N - Boc - 3 - amino - 3 - phenylpropanoic acid was resuspended in 5:2 (v/v) CH 2 Cl 2 : THF (dry, 10 mL ) and stirred under nitrogen. Triethylamine (TEA) was added (1.2 equiv) and stirred at 22 °C for 10 min. The carboxylate activating reagent, ethyl chloroformate (1.2 equiv) was added dropwise, and the reaction was monitored by TLC (1% acetic acid in diethy l ether) for 2 h. Solvents were evaporated to dryness under a nitrogen stream. The mixed anhydride product was resuspended in t - butanol (0.5 mL) and stirred. CoA (1 equiv) (dissolved in 1 mL of 0.4 M NaHCO 3 ) was added to the mixed anhydride intermediate an d the reaction was monitored by TLC (5:3:2 (v/v/v) 1 - butanol, water, acetic acid) for up to 4 h. The reaction was stopped by the addition of 1M HCl (pH 4). The identity of the acyl CoA product was further verified by LC - ESI - MS . Without further purificatio n, the R - N - Boc - 3 - amino - 3 - phenylpropanoyl CoA was stirred on ice at 0 °C. TFA was added dropwise to a final concentration of 30% (v/v) and stirred for 30 min. The reaction was warmed to room temperature and stirred for 1 h, followed by 1 h on ice. The flask alternated between stirring on ice for 1 h and at room temperature for 1 h each for 3 cycles. The reaction progress was followed by TLC (5:3:2 (v/v/v) 1 - butanol, water, acetic acid) and LC - ESI - MS. The crude product was back - extracted with ethyl acetate (3 x 15 mL) to remove t - butanol and organic contaminants. 166 The remaining aqueous phase (< pH 4) was dried under vacuum to remove excess TFA and then lyophilized. (3 R ) - - phenylalanyl CoA was resuspended in water (pH 5, 0.5 mL) and purified by C18 column chromatography with a mobile phase of 0.05% TEA (aq.) with 2.5% acetonitrile. Fractions containing the product were pooled, desalted by running through Dowex 50 cation ex change resin, flash - frozen, and lyophilized. The (3 R ) - - phenylalanyl CoA was characterized by NMR ( Figure A.3.2 ), UV - HPLC (Figure A.3.3 ), LC - ESI - MS ( Figure A.3.4 ), and the Ellman assay. 1 H - NMR (500 MHz, D 2 O) (ppm): 0.76 (br s, H11 ), 0.9 (br s, H10 ), 2.93 (m, H9 ), 3.22 (m, H2c), 3.29 - 3.46 (m, H5 , H8 ), 3.55 (q, H5 ), 3.81 (m, H5 ), 4.01 (s, H3 ), 4.20 (br s, H1 ), 4.54 (br s., H4 ), 4.81 - 4.85 (m, H2 , H3 , H3c), 5.07 (m, NH 2 ), 6.15 (m, 1 ), 7.38 (aromatic H), 8.36 (br, s, adenine H), 8.6 (m , adenine H); ( Figure A.3.2 for proton numbering). LC - ESI - MS m / z 913.1428 [M - H] - ; calculated for C 30 H 44 N 8 O 17 P 3 S: 913.1736. 4.2.3 Method I. Synthesis of (2R,3S) - phenylisoserinyl CoA N - Boc protection of (2 R ,3 S ) - phenylisoserine was performed in the same manner as f or (3 R ) - - phenylisoserine, however this reaction was conducted in small scale (<10 mg, 98% yield) due to limited substrate availability. Mixed anhydride formation with ethyl chloroformate and thioesterification with CoA were performed as described for the synthesi s of (3 R ) - - phenylalanyl CoA. N - Boc protected intermediate (~9.8 mg, 98 % yield, 95% purity) was analyzed by NMR. 1 H - NMR (500 MHz, CDCl 3 ) (ppm): 1.43 (m, H5), 2.88 (m, H2), 5.12 (br. d., H3), 7.30 7.36 (aromatic protons). (See Figure A.3. 10 for proton numbering). 167 4.2.4 Method II. Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA N - Boc protection of ( 2 R ,3 S ) - phenylisoserine (98% yield) at smallscale (<5 mg) was performed as described above. The hydroxyl group of N - Boc - (2 R ,3 S ) - phenylisoserine was protected with tert - but yldimethylsilyl chloride (TBDMS - Cl). Briefly, N - Boc - (2 R ,3 S ) - phenylisoserine (16 µmol) was dissolved in DMF (0.2 mL) and stirred with molecular sieves (4 Å) to remove residual water. Imidazole (2.5 equiv) was added to the solution and stirred (~30 min). TBD MS - Cl (1.2 equiv) was then added and allowed to stir for 18 h. If remaining starting material was visible by TLC, more TBDMS - Cl (0.1 equiv) was added. The product was extracted twice with diethyl ether, then washed with brine to remove residual DMF. After removal of diethyl ether by evaporation, some residual DMF remained and converted yields were estimated to be 80 - 90% based on TLC. The identity of the product was confirmed by LC - ESI - MS. Without further purification, the mixed anhydride was made with ethyl chloroformate as described above and thiolation by CoA (0.8 equiv) was also performed as previously described. This method was not successful and did not yield any detectable product by the standard methods of analysis used earlier to characterize (3 R ) - - Phe CoA. Therefore, this method was abandoned in favor of Method III. 4.2.5 Method III. Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA N - Boc protection of ( 2 R ,3 S ) - phenylisoserine (98% yield) at smallscale (<5 mg) was performed as described above. Without protection o f the 2 - hydroxyl group, carboxylate activation was attempted by N,N' - dicyclohexylcarbodiimide ( DCC)/ hydroxybenzotriazole (HOBt) coupling in small scale (<5 mg). Briefly, N - Boc - (2 R ,3 S ) - phenylisoserine (25.6 µmol) was added to a round bottom flask, dissolve d in 500 µL of 5:2 (v/v) dry 168 THF:CH 2 Cl 2 , and stirred for 10 min at 22 °C. TEA (1 equiv) was added and stirred for an additional 10 min. This solution was then added dropwise to a stirred solution of DCC (1.1 equiv) and the reaction progress was monitored b y TLC. The HOBt (1.1 equiv) was pre - incubated with TEA (1 equiv) and solubilized in DMF before dropwise addition to the DCC coupling reaction. The reaction was stirred under nitrogen for 18 h at 22 °C and monitored by TLC (1:9 MeOH: CHCl 3 (v/v)). The react ion was filtered, dried under nitrogen and redissolved in t - butanol (0.5 mL). CoA (1 equiv) was dissolved in 0.4 M Na 2 HCO 3 (0.5 mL). Portions of CoA (50 µL) were added dropwise to the activated intermediate every 10 min. After addition of CoA, the reaction continued for an additional ~3 h and was monitored by TLC (5:3:2 n - butanol: water: acetic acid (v/v/v)). This method consistently yielded a maximum of detectable product (< 5%) by LC - ESI - MS. Therefore, this method was abandoned in favor of Method IV. 4.2.6 Meth od IV. Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA The method of DCC/HOBt coupling was modified to DCC/NHS ( N - hydroxysuccinimide) coupling. The reaction was tested in small scale (~3.55 µmol) as described above for DCC/HOBt coupling except NHS was added in p lace of HOBt. The thioesterification reaction was modified by dissolving CoA in degassed 0.4 M Na 2 HCO 3 with 1 mM tris - (2 - carboxyethyl) phosphine (TCEP). The activated (2 R ,3 S ) - phenylisoserinyl intermediate was added dropwise to the stirring solution of CoA over 30 min and allowed to run for 5 h. This method consistently yielded a maximum of detectable product (<5%) by LC - ESI - MS. Therefore, this method was abandoned in favor of a biosynthetic method. 169 4.2.7 PheAT purification PheAT was expressed in BL21(DE3) E. coli (Invitrogen, Carlsbad, CA) ( 48 ) . A single colony was selected and used to inoculate a 100 - mL culture of LB media containing 50 µg/mL kanamycin. The culture was grown overnight at 37 °C. Aliquots of this starter culture (7 mL) were used to inoculate a flas k of fresh LB media (1 L) containing 50 µg/mL kanamycin. For large preparations of PheAT, 5 to 10 L were grown at once. This culture was grown at 37 °C until A 600 = 0.6. Gene expression was induced by 0.2 mM IPTG, grown overnight (~16 h) at 18 °C, and cell s harvested by centrifugation at 8,000 g for 5 min. Pellets were resuspended in Buffer A (50 mM HEPES, 300 mM NaCl, 15 mM imidazole, 5% glycerol, pH 8.0) at 3 mL/g pellet (wet weight) with PMSF (1 mM). Cells were kept on ice, lysed with a Misonix XL 2020 so nicator and centrifuged at 18,000 g for 20 min. The supernatant was further centrifuged in a Beckman Coulter Ultra Centrifuge at ~100000 g for 1.5 h. The supernatant was loaded onto a Ni 2+ NTA Qiagen column pre - equilibrated with Buffer A at 4 °C. The column was washed with 5 column volumes (CV) of Buffer A, and eluted with 3 CV of Buffer A containing 250 mM imidazole. The eluent was concentrated in a Millipore Amicon Ultra 30 kDa cutoff concentrator and buffer exchanged with 50 mM HEPES, pH 8.0. Final concent rations were ~50 mg/mL (estimated by the Coomassie (Bradford) Protein Assays (Thermo Scientific Pierce, Grand Island, NY)). Protein aliquots were flash - frozen in liquid nitrogen and stored at 80 °C. 4.2.8 Biosynthesis of (2 R ,3 S ) - phenylisoserinyl CoA A large - sc ale preparative PheAT enzymatic assay was performed under the following conditions. A concentrated solution of PheAT (20 mL of 36 mg/mL) in 10 mM HEPES buffer, pH 8 was gently stirred at 23 °C. MgCl 2 (6H 2 O) (100 mg) and (2 R ,3 S ) - 170 phenylisoserine (0.17 mmol) were dissolved in the PheAT solution. Separately, ATP (0.17 mmol) and CoA (0.13 mmol) were dissolved in 2 mL each of 10 mM HEPES, pH 8. Solutions of ATP and CoA were adjusted to pH 8 (0.5 M NaOH) before addition to the stirred PheAT solution. Total reactio n volume was 20 mL. Reaction progress was monitored by UV - HPLC. Specifically, the peaks corresponding to CoA (4.7 min) and (2 R ,3 S ) - phenylisoserinyl CoA (6.6 min) were monitored. After 7.5 h, 50% of the CoASH was converted to product and the reaction was st opped by addition of 8.8% formic acid to pH 4 to precipitate PheAT. The precipitated reaction was centrifuged at 5000 g for 10 min. The supernatant was collected, the pellet washed with water (pH 4 with formic acid) and re - centrifuged. Supernatants were com bined and filtered through a Millipore Amicon Ultra 30 kDa concentration unit to remove trace protein. The flow - through was collected, flash - frozen in liquid nitrogen, and lyophilized. The lyophilized crude product was then dissolved in 2 mL of ultrapure w ater (pH 4), for preparative HPLC purification. 4.2.9 Purification of (2 R ,3 S ) - phenylisoserinyl CoA (2 R ,3 S ) - phenylisoserinyl CoA (PhIS - CoA) residue was resuspended in 0.1% TFA in water and purified by preparative HPLC (Agilent 1100 Series). Approximately 20 aliq uots (100 µL) of the filtered, lyophilized, crude mixture were loaded onto a 100 µL loop and purified on a preparative C18 column (Atlantis C18 OBD, 5 µm, 19 mm × 150 mm). The column was eluted at 4 mL/min with 2.5% solvent B (100% acetonitrile) and 97.5% solvent A (0.1% trifluoroacetic acid in water) with a 4 min hold, a linear gradient to 30% solvent B over 15 min, then increased to 100% solvent B over 2 min, and finally lowered to 2.5% solvent B over 2 min. The effluent was monitored at 258 nm. Peak frac tions were collected, flash - frozen, and lyophilized to yield (2 R ,3 S ) - phenylisoserinyl CoA (97.5% pure, 45.5 mg 171 (0.05 mmol) at 30% yield relative to (2 R ,3 S ) - phenylisoserine. The purified thioester product was analyzed by NMR (Figure A.3. 11 , Figure A.3.12 ), UV - (Figure A.3.13 ), mass spectrometry ( Figure A.3.14 ) and Ellman assay for free thiol. 1 H NMR (500 MHz, D 2 O, pH 3 with CD 3 COOD) : 0.59 (s, 11 ), 0.73 (s, 10 ), 2.16 (br s, 6 ), 2.72 (br s, 9 ), 2.95 3.30 (m, 5 , 8 ), 3.37 (m, 5 ) , 3.64 (m, 5 ), 3.85 (s, 3 ), 4.02 (br s, 1 ), 4.38 (d, J = 7.32 Hz, 2c), 4.46 (d, J = 6.35 Hz, 4 ), 4.74 (m, 2 , 3 , 3c), 4.89 (br s, NH 2 ), 5.99 (d, J H). (See Figure A.3. 11 for proton numbering) 13 C NMR (126 MHz, D 2 O) : 20.48 (s, 11 ), 23.17 (s, 10 ), 30.00 (s, 9 ), 37.57 (s, 5 , 6 ), 40.43 (s, 8 ), 40.66 (d, J = 7.67 Hz, 2 ), 59.17 (s, 3c), 67.30 (br s, 5 ), 74.09 (s, 1 ), 76.32 (m, 2 , 3 , 3 ), 79.24 (s, 2c), 89.60 (br s, 1 , 4 ), 120.78 (s, 5), 129.87 - 134.79 (C4 aromatic carbons), 144.64 (s, 8), 146.91 (s, 4), 150.74 (s, 2), 152.15 (s, 6), 176.33 (s, 7 ), 176.98 (s, 4 ), 205.43 (s, 1c). (See Figure A.3. 12 for carbon numbering). LC - ESI - MS m / z 929.094 [M H] - ; cal culated for C 30 H 44 N 8 O 18 P 3 S: 929.1713. 4.2.10 HPLC Analysis of acyl CoA thioesters Acyl CoA synthesis and purity were evaluated by HPLC. Samples were dissolved or diluted in Milli - Q water, pH 4 and injected (10 µL) onto a C18 column using an Agilent 1100 Series HP LC system. HPLC was used to determine the purity of the biosynthetic acyl CoA products. An autosampler connected to an HPLC system (Agilent 1100) injected a 10 - µL sample onto a C18 HPLC column (3.5 µm, 4.6 mm × 100 mm, at 22 °C, Waters). The column was elu ted at 1 mL/min with 2.5% solvent B (100% acetonitrile) and 97.5% solvent A (0.1% trifluoroacetic acid in water) with a 1 min hold, a linear gradient to 30% solvent B over 10 min, then increased to 100% solvent B over 2 min, and finally lowered to 2.5% sol vent B over 2 min. The effluent was monitored at A 258 . Re action progress was 172 measured by the disappearance of the peak corresponding to CoASH and the growth of the peak corresponding to the acyl CoA of interest. Peak areas were integrated using Agilent Che mStation (Agilent Technologies, Inc. 2003, Santa Clara, CA) software to determine the extent of the reaction or the purity of the acyl CoA. 4.2.11 Acyl CoA purity analysis (Ellman assay) 5,5' Dithio bis (2 nitrobenzoic acid ): DTNB ) is applied i n a well - established method to quantify free thiol concentration in protein solutions or purified acyl CoA thioesters ( 61, 62 ) in a sample of purified acyl CoA that contains salt. Free th were adapted ( 60 ) as follows. Briefly, DTNB was dissolved in 30 mM MOPS, pH 7 to make a 10 mM stock solution. An aliquot of the 10 mM DTNB (50 µL) stock was diluted (2.5 mL) with buffer, then mixed with acyl CoA (<0.2 mM) ( 250 µL) dissolved in the same buffer. The reactants were incubated at room temperature for 10 min before measuring the absorbance at 412 nm on a UV/VIS spectrophotometer. Acyl CoA thioesters began to hydrolyze at longer incubation times, resulting in incre asing A 412 . DTNB = 14.15 mM - 1 cm - 1 at 412 nm) was used to determine the amount of free thiol present in the sample. This number was compared to a control sample; mixed with NaOH (0.5 M final concentration), heated 20 min (55 °C), acidified to pH 7 (1 M HC l) and mixed with the diluted DTNB (as described), and incubated for 20 min at 22 °C. This sample represented the amount of free thiol and acyl CoA from the absorbanc es of both hydrolyzed and non - hydrolyzed samples. The amount of salt in the purified acyl CoA material was determined by calculating the total mass of CoA and acyl CoA present from moles as determined by 173 Ellman assays. This mass was substracted from the to tal mass of purified material to give the amount of salt (non - acyl CoA) material present. 4.2.12 BAPT, BadA, and NDTNBT purification BAPT was purified as MBP - BAPT as described in Chapter 3 of this dissertation. BadA was purified as described in Chapter 2 of this dissertation. NDTNBT was purified as previously described ( 55 ) . 4.2.13 BAPT kinetic assays Solutions of each acyl CoA (10 mM) were dissolved in deionized water, pH 3 (8.8% formic acid). Baccatin III (10 mM) and 10 - deacetylbaccatin III (10 mM) were dissolved in a cetonitrile or methanol, respectively. To establish steady - state kinetic rates of MBP - BAPT with respect to enzyme concentration and time, the acyl CoA and taxane were combined in 200 mM MOPS,(pH 8) containing 5% glycerol and pre - incubated at 31 °C for 10 m in before the addition of ~0.2 µg (0.024 nmol) MBP - BAPT (200 µL total volume). Assays were acid - quenched with 8.8% formic acid (aq.) to pH 5 at different timepoints to generate a time - course series. Docetaxel (1 µM) was added as an internal standard. Conce ntrations of acetonitrile or methanol (depending upon the taxane substrate) were held constant at 10% (v/v). Assays were then extracted twice with 1 mL of EtOAc and dried under nitrogen and/or vacuum. Dried assays were resuspended in 100 µL of acetonitrile (baccatin III - containing assays) or methanol (10 - deacetylbaccatin III containing assays) and transferred to a GC vial (200 µL insert) for analysis by LC - ESI - MS/MS. 174 4.2.14 Kinetic analysis To calculate kinetic constants, each substrate was varied (1 3000 under the predetermined steady - state conditions. The products made in the reaction assays were quantified as described above. Kinetic parameters ( K M and k cat ) were calculated by non - linear regression with Origin 9.0 using the follow ing equation v o = k cat [E o ][S]/( K M + [S]) (Northampton, MA) ( Figure s A.3.1 5 to A.3.17 ) . Although K M is not a true dissociation constant, in this study it will serve as a means of comparing enzyme interactions with both substrates. Assays were prepared as f ollows. The taxane was added to a 5 mL glass test tube, 200 mM MOPS (pH 8) was added dropwise to facilitate mixing of the acetonitrile or methanol. The reaction tube was incubated at 31 °C for 10 min, before the acyl CoA and MBP - BAPT (0.023 nmol in 200 µL) were added to start the assay. 4.2.15 Liquid chromatography mass spectrometry: BAPT assay analysis LC - ESI/MS/MS in positive ion mode was used to quantify the biosynthetic acyl CoA products. An autosampler (at 10 °C) connected to an HPLC system (Waters Corp., Mi lford, MA) injected 10 - µL of each processed assay onto an Ascentis Express C18 HPLC column (2.7 µm, 5 cm × 2.1 mm, at 30 °C, Sigma - Aldrich). Assay products dissolved in acetonitrile or methanol were each analyzed with an HPLC method utilizing acetonitrile or methanol as the mobile phase, respectively. The column was eluted at 0.4 mL/min with 2.5% solvent B (100% methanol) and 97.5% solvent A (0.5% formic acid in water) with a 0.5 min hold, an immediate increase to 30% solvent B, followed by a linear gradien t to 90% solvent B over 4 min, then increased to 100% solvent B over 0.5 min, and finally lowered to 2.5% solvent B over 0.5 min. The needle was washed with 0.4 mL each of 100% isopropanol and then with 10% acetonitrile in water prior to each injection. Th e HPLC effluent was 175 directed to an electrospray ionization mass spectrometer (Quattro Micro, Waters Corp, Milford, MA) in positive ion mode, with a cone voltage of 20 V and collision energy of 16 eV. Each taxane was quantified by multiple reaction monitori ng of the [M + H] + m / z 509 transition, common to both baccatin III, 10 - DAB, and docetaxel. Peak areas (calculated using the MassLynx data analysis software, Waters Corp., Milford, MA) were converted to product concentrations using the docetaxel internal standard of 1 µM). 4.2.16 PheAT and BAPT coupled reactions Coupled assays for the production of N - debenzoyl - - deoxypaclitaxel were prepared on ice and incubated at 31 °C for 5 min before the enzyme was added. Assay volume was 200 µL with 10% ( v / v ) methanol. Baccatin III (0.75 mM t o 3 mM dissolved in methanol), was added first to a 5 - mL glass test tube, followed by 200 mM MOPS, (pH 8), 3.5 mM MgCl 2 , 1 mM ATP, 1 mM phenylisoserine, 0.05 to 2 mM CoA, and PheAT (0.75 mg/mL) to a final volume of 200 µL. After 1 h at 31 °C, MBP - BAPT (0.0 1 mg/mL) was added and allowed to react for an additional two h. Assays were processed for analysis in the same manner as that for the MBP - BAPT kinetics (see above). Coupled assay products were analyzed by LC - ESI - MS/MS with multiple reaction monitoring of the N - debenzoylpaclitaxel ion [M + H] + m / z m / z 509 transition and of the internal standard docetaxel (1 µM) ion [M + H] + m / z m / z 509 transition. 4.2.17 Production of paclitaxel and its analog s in a coupled enzyme assay Four enzymes (PheAT, BAPT, BadA, and NDTNBT) were coupled together to produce paclitaxel (or an N - acyl analog ) from the precursor baccatin III. The coupled reaction was tested in vitro where the ligase reactions, catalyzed by PheAT and BadA, were done first, followed by addition of the a cyltransferases BAPT and NDTNBT. The ligase reactions 176 were prepared on ice and incubated, shaken (100 rpm) in a 31 °C waterbath for 5 min prior to the addition of PheAT and BadA. Baccatin III (0.5 mM dissolved in methanol) was added to the bottom of a glas s test tube and mixed with 200 mM MOPS, pH 8. Substrates were then added to final concentrations of 3.5 mM MgCl 2 , 1 mM ATP, 50 µM to 1 mM CoA, 0.5 to 2 mM (2 R ,3 S ) - phenylisoserine, and 1 mM benzoic acid (or 2 - furoic acid). BadA (1 µg/mL) and PheAT (~1 mg/mL ) were then added to start the assay after temperature equilibration. After 1 h, MBP - BAPT (2.3 µg, 0.023 nmol) and NDTNBT (91 µg, 1.7 nmol) were added to a final volume of 250 µL and incubated for 3 h. Docetaxel (1 µM) was added as an internal standard and the solution was extracted with ethyl acetate (2 × 1 mL). The organic fraction was removed, dried under a stream of nitrogen, and resuspended in 100 µL of methanol. Transition ions for the production of paclitaxel ([M + H] + = m / z m / z 509), N - debenzo ylpaclitaxel ([M + H] + = m / z m / z 509), and N - debenzoyl - N - 2 - furanylpaclitaxel ([M + H] + = m / z m / z 509), were analyzed by LC - ESI - MS/MS using the same methods for determining MBP - BAPT kinetics described before. Product concentrations were normali zed relative to the internal standard of docetaxel (1 µM). 177 4.3 Results and d iscussion 4.3.1 Synthesis of acyl CoA substrates Characterization of BAPT acyltransferase activity requires acyl CoA substrates that are not commercially available. Therefore, the substrat es (3 R ) - - phenylalanyl CoA ((3 R) - Phe CoA) and (2 R ,3 S ) - phenylisoserinyl CoA ((2 R ,3 S ) - PhIS CoA) were synthesized and biosynthesized, respectively ( Figure 4 . 5 ). Several methods have been developed for acyl CoA synthesis inv olving thioester activation to a mixed anhydride or other activated intermediate with ethyl chloroformate, N , N - carbonyldiimidazole, N - hydroxysuccinimide or others ( 58, 60, 63 - 67 ) . While synthesis of the (3 R ) - - Phe CoA was relatively straightforward, synth esis of (2 R ,3 S) - PhIS CoA proved more complicated, ultimately requiring a large - scale biosynthetic approach with a repurposed bacterial adenylation domain (PheAT) from tyrocidine synthetase A (TycA) of Bacillus brevis ( 42, 48, 68, 69 ) . Figure 4 . 5 . Structures of (3 R ) - - phenylalanyl coenzyme A (R = H) and (2 R ,3 S ) - phenylisoserinyl coenzyme A (R = OH). 178 4.3.1.1 Synthesis of (3R) - - phenylalanyl CoA A method based on the mixed anhydride intermediate was used to synthesize (3 R ) - - Phe CoA, ( Figure 4.6 ) ( 43, 67 ) . The amino group of (3 R ) - - phenylalanine was N - Boc protected, followed by the addition of ethyl chloroformate to produce a mixed anhydride intermediate. Incubation with CoA and subsequent deprotection of the 3 - amino group yielded (3 R ) - - Phe CoA. Characterization of the pur HPLC ( Figure A.3.3 ), mass spectrometry ( Figure A.3.4 ), and NMR ( Figure A.3.2 ). Results and ~95% purity (compared t o free thiol content) with 50% salt by weight. Problems encountered during synthesis of (3 R ) - - Phe CoA were degradation during purification, lyophilization, and high salt. These problems were minimized with desalting resin and by shortening lyophilization time with smaller volumes. The source of retained salt was likely the TEA used during colum n purification. The (3 R ) - - Phe CoA is a zwitterion at pH 5, and likely purified as a salt even after desalting. NMR spectrum analysis agrees with prior syntheses of related compounds ( Figure A.3.2 and Figure A.3.3 ) ( 70 - 72 ) . The presence of smaller, shifted peaks in NMR spectra is also observed in the stock material of CoA from multiple sources and are possibly from an isomer of CoA ( 73 ) . Mass spectrometry analysis of the major purified product was consistent with the expected mass of (3 R ) - - Phe CoA, m / z 913 ( Figure A.3.4 ). Tandem mass spectrometry analysis of a BAPT assay with purified (3 R ) - - Phe CoA with baccatin III and showed a mass consistent with N - debenzoyl - - deoxypaclitaxel ( Figure A.3.5 ). 179 Figure 4 . 6 . Organic synthesis of (3 R ) - - Phenylalanyl CoA. 4.3.1.2 Synthesis of (2R,3S) - Ph enylisoserinyl CoA Synthesis of (2 R ,3 S ) - PhIS CoA was more complicated than (3 R ) - - Phe CoA. Several different synthetic approaches were employed, but resulted in very little (<1 - 10%) or no yield. The 2 - hydroxyl group on phenylisoserine changes the reactivity of the substrate with activating agents such as ethyl chloroformate. In the first synthetic attempt the 2 - hydroxyl position of the phenylisoserine was not protected in order to reduce steric hindrance at the carboxylate reaction center (see Method I in Materials and Methods , Figure A.3.6 ). The carboxylate was proposed to react in sequence with ethyl chloroformate to form the mixed anhydride intermedi ate and finally with CoA to form the acyl CoA thioester. Using the same synthetic method to make (2 R ,3 S ) - PhIS CoA as used with (3 R ) - - Phe CoA, resulted in the production of many side products as observed on TLC and by mass spectrometry. The side products a ppeared comprised of the desired (2 R ,3 S ) - PhIS CoA in trace amounts and numerous derivatives of the N - Boc - (2 R ,3 S ) - phenylisoserine mixed anhydride. Attempts to modulate cross - reactivity by cooling the reaction on ice, or slowly adding cold, dilute ethyl chlo roformate were unsuccessful. 180 The persistent production of by - products in the mixed anhydride method described - - hydroxyl protecting group for this synthesis needed to be small and acid labile at room temperature , to prevent cleavage of the acyl CoA thioester product. The 2 - hydroxyl group was protected as a t - butyldimethylsilyl ether (TBDMS ether) ( 74 ) . This caused an increase from two to five h in incubation time to form the mixed anhydride intermediate compared with (3 R ) - - phenylalanine (Figure A.3.7) . After reacting the mixed anhydride intermediate with CoA, we observed analytes with mass fragments consistent with N - Boc - (2 R ,3 S ) - PhIS CoA. However, a bulk of the product mixture contained unreacted CoA which likely formed dimers as evidenced by an ion at [CoAS - SCoA - 2H] - 2 = m / z 765.10 and isotopic peaks separated by 0.5 m / z units (not shown). The low conversion of the TBDMS - protected intermediate was likely caused by steric hindrance that prevented CoA from accessing the re active carbonyl group. For this reason, an alternative method of carboxylate activation was sought. After the initial ethylchloroformate approach resulted in extensive byproducts and protection of the 2 - hydroxyl group resulted in trace product formation, an alternative to carboxylate activation that prevents the formation of byproducts was designed. The dicyclohexylcarbodiimide (DCC) reagent is well - established in organic chemistry as a means of activating carboxylic acids in peptide coupling chemistry ( 75 , 76 ) . The large size of DCC was predicted to reduce the occurrence of side products through steric hindrance with the 2 - hydroxyl protecting group. However, the bulkiness of DCC would also likely be a steric hindrance for the final nucleophilic reaction wi th CoA. Therefore, DCC was coupled to hydroxybenzotriazole (HOBt), a small, planar leaving group ( Figure A.3.8 ) 181 ( 75 ) . The activation by HOBt was followed by TLC and by observing the precipitation of the insoluble DCC byproduct dicyclohexylurea (DCU). There was no N - Boc - (2 R ,3 S ) - phenylisoserinyl CoA detectable above background levels by mass spectrometry ([M - H] - 1 = m / z 1029.22), substantial CoA oxidation was observed, and the activated intermediate appeared to be degraded into phenylisoserine and HOBt. The D CC/HOBT coupling method was modified to include N - hydroxysuccinimide (NHS) instead of HOBt ( Figure A.3.9 ) ( 75 ) . The NHS reagent is more polar than HOBt and the resulting mixed anhydride with (2 R ,3 S ) - PhIS was hypothesized to be more water soluble and possib ly more reactive with CoA. The non - sulfur reducing agent tris - (2 - carboxyethyl) - phosphine (TCEP) was also added to prevent CoA dimerization. Upon CoA addition, results were similar with no significant N - Boc - (2 R ,3 S ) - phenylisoserinyl CoA observed. However the CoA was not oxidized due to inclusion of the reducing agent, TCEP. Figure 4 . 7 . Conformation of (2 R ,3 S ) - phenylisoserine. A. Linear representation of (2 R ,3 S ) - phenylisoserine. The bond highlighted in blue is selected for the Newman projection. B. Newman projection of (2 R ,3 S ) - phenylisoserine. Structure was energy minimized in ChemBio 3D Ultra (Perkin Elmer, Waltham, MA). In general, the failure to obtain appreciable quantities of N - Boc - (2 R ,3 S ) - phenylisoserinyl CoA by all synthetic means tested is indicative of a sterically challenged reaction center ( Figure 4 . 7 ). Substantial CoA dimerization was observed in these reactions, 182 but not in t he (3 R ) - - Phe CoA synthetic reaction, suggesting CoA reacted with itself because the reactive intermediate was not accessible. These difficulties made it apparent that a different method for synthesizing (2 R ,3 S ) - PhIS CoA in large enough quantities (~50 - 100 mg) w as needed for the kinetic characterization of BAPT. Figure 4 . 8 . Biosynthesis of (2 R ,3 S ) - phenylisoserinyl CoA with PheAT, a truncated form of tyrocidine synthetase A (TycA). - hydroxyl group is shown in red. 4.3.2 Biosynthesis of (2 R ,3 S ) - PhIS CoA. Considering the numerous challenges encountered with the organic synthesis of (2 R ,3 S ) - PhIS CoA, this approach was abandoned, and a biosynthetic method was considered. Recently in th e Walker group, the TycA (PheATE) monomer from the non - ribosomal peptide synthase (NRPS) tyrocidine synthetase A biosynthetic pathway was tested as a CoA ligase and showed broader substrate activity with (3 R ) - - phenylalanine and (2 R ,3 S ) - phenylisoserine bey - phenylalanine ( 42, 68, 69 ) . Modified TycA activity produces (3 R ) - - Phe CoA and (2 R ,3 S ) - PhIS CoA in an ATP dependent manner. To our knowledge it is the only enzyme currently known to produce (2 R ,3 S ) - PhIS CoA. More recently, a tr Figure 4 . 8 ). The truncation was active and is hereafter referred to as PheAT ( 48 ) . The Michaelis - Menten kinetic parame ters of PheAT ( k cat = 0.015 ± 0.0028 s - 1 and K M = 440 ± 62 µM for (2 R ,3 S ) - PhIS, and K M = 208 ± 57 µM for CoA) were used to 183 Figure 4 . 9 . Biosynthetic reaction progress curve for (2 R ,3 S ) - PhIS CoA . A large - sc ale (20 mL) reaction with PheAT (36 mg/mL), CoA (5 mg/mL), ATP (5 mg/mL), (2 R ,3 S ) - PhIS (~1.6 mg/mL) and MgCl 2 (5 mg/mL) was monitored by UV - HPLC at 258 nm. The peak corresponding to (2 R ,3 S ) - PhIS CoA was normalized to % conversion relative to CoA. Black cir cles: One PheAT large - scale reaction which was supplemented with additional (2 R ,3 S ) - PhIS (0.5 mM) at 7 h (red squares). Black triangles: One PheAT large - scale reaction, which was supplemented with additional ATP (5 mM) at 6 h (blue diamond). estimate ~70 0 mg of PheAT is needed to produce ~100 mg of (2 R ,3 S ) - PhIS CoA in 1 h ( Table 4 . 2 ). PheAT was heterologously expressed as a 6X - His tagged fusion protein from E. coli and purified by nickel - affinity chromatography at 99% purity. PheAT was highly soluble at 70 mg/mL. The reaction progress of a large - scale (20 mL) enzyme reaction was monitored by HPLC over the course of 6 to 19 h (see Methods) ( Figure 4 . 9 ). Accumulation of (2 R ,3 S ) - PhIS CoA never exceeded 60% ( Figure 4 . 9 ) perhaps because of a gradual loss of PheAT activity, or because the reaction reached an equilibrium between (2 R ,3 S ) - PhIS CoA production and degradation in the buffer at p H 8.0. Upon quenching the reaction by acidification (pH < 5.0 with 8.8% formic acid) and stabilizing (2 R ,3 S ) - PhIS CoA, PheAT also precipitated, facilitating subsequent product purification. 184 Separation of (2 R ,3 S ) - PhIS CoA from CoA was a concern during biosy nthetic production. The two compounds had similar retention factors (R f values) with all TLC conditions tested including variations on 5:3:2 butanol:water:acetic acid (v/v/v) which was successful at separating (3 R ) - Phe CoA from CoA. Therefore, preparative, reverse - phase HPLC was used for purification to obtain 45.5 mg (50 µmol) in 30% yield (relative to (2 R ,3 S ) - phenylisoserine) at 97% purity by UV - HPLC analysis ( Figure A.3. 13 and Figure A.3.14 ). Previous (2 R ,3 S ) - PhIS CoA synthesis isolated 2.9 µmol in 10% y ield (80% purity) ( 43 ) . This preparative purification of (2 R ,3 S ) - PhIS CoA is an improvement over synthetic methods makes it possible to characterize the paclitaxel biosynthetic BAPT enzyme for the first time ( Figure 4 . 10 ). Figure 4 . 10 . BAPT - catalyzed enzyme reaction. The proposed native BAPT reaction catalyzes the transfer of (3 R ) - - phenylalanyl CoA (R 2 = H) to the 13 - O of baccatin III (R 1 = Acetyl group). BAPT is less active with (2 R ,3 S ) - phenylisoserinyl CoA (R 2 = OH) when incubated with either baccatin III or 10 - DAB (R 1 = H). 4.3.3 Michaelis - Menten kinetics of BAPT 4.3.3.1 (3 R ) - - phenylalany l CoA and baccatin III substrates The importance of the phenylisoserinyl side chain for the efficacy of paclitaxel has been well - established in structure and function studies ( 57, 77 ) . Current literature supports a biosynthetic pathway in which BAPT cataly zes the C13 acylation of baccatin III with (3 R ) - 185 - phenylalanyl CoA to form N - debenzoyl - - deoxypaclitaxel (ND2DP), which then - position on the sidechain, and N - benzoylated to produce paclitaxel ( 43, 56, 78 ) - - PL) was identifi ed in Taxus baccata cell cultures elicited with jasmonic acid ( 20 ) - - phenylalanine and 4 - - PhL with phenylisoserines was not characterized in the former study. While the discovery of a Ta xus - PhL is long - - hydroxyl group to ND2DP remains elusive. In our efforts to characterize the activity of BAPT and build the foundation for engineering efforts of paclitaxel biosynthesis from baccatin III, both the synthetic (3 R ) - - Phe CoA and the biosynthetic (2 R ,3 S ) - PhIS CoA were utilized as substrates. Michaelis - Menten constants for BAPT were determined after (bio)synthesizing the needed acyl CoA substrates ( Table 4 . 2 ). More importantly, this is the first report of the kinetic parameters for BAPT with (2 R ,3 S ) - PhIS CoA. The k cat and K M of BAPT for (3 R ) - - Phe CoA with baccatin III at apparent saturation were 0.0583 ± 0.0010 s - 1 and 5.6 ± 1.0 µM, respectively ( Fi gure A.3.16 ). Reciprocally, the k cat and K M of BAPT for baccatin III with (3 R ) - - Phe CoA at apparent saturation were k cat = 0.0575 ± 0.0018 s - 1 and a 68 ± 9.6 µM, respectively ( Figure A.3.17 ). These values are compared with those of BAPT with (2 R ,3 S ) - PhIS CoA where k cat = 0.0022 ± 0.00013 s - 1 ( Figure A.3.15 ) and K M = 22.4 ± 6.9 µM. Previous estimates of the K M of BAPT using crude lysate from a recombinant BAPT expressed in E. coli , were 2.4 ± 0.5 µM and 4.9 ± 0.3 µM for baccatin III and (3 R ) - - Phe CoA, res pectively ( 43 ) . These estimates different than those calculated with purified BAPT. In this study, the K M of purified BAPT with baccatin III is 12 - times higher than that for (3 R ) - - Phe CoA, whereas the earlier report determined a K M that is half that for ( 3 R ) - 186 - Phe CoA. These disparities likely result from the use of crude enzyme preparations of recombinant enzyme expressed without an affinity epitope for purification and subsequent quantification in the earlier study ( 43 ) . In this study, we expressed BAPT with a polyhistidine affinity epitope tag, purified, and quantified the expressed enzyme with greater accuracy compared with the crude estimates used previously ( 43 ) . 4.3.3.2 (2R,3S) - Phenylisoserinyl CoA and baccatin III substrates - hydroxyl group on paclitaxel is required for its biological activity. Thus characterization of BAPT enzymatic activity for (2 R ,3 S ) - PhIS CoA will help advance bioengineering efforts toward making paclitaxel and likely its analog s. BAPT is 27 - times more active with (3 R ) - - Phe CoA than with (2 R ,3 S ) - PhIS CoA, (with baccatin III at apparent saturation) which suggests that the proposed paclitaxel biosynthetic pathway may - - hydroxyl of (2 R ,3 S ) - PhIS CoA increases the K M 4 - fold over that of (3 R ) - - Phe CoA. This may be due to unfavorable steric or electronic effects of the added hydroxyl group within the active site. 4.3.3.3 ( 2 R ,3 S ) - Phenylisoserinyl CoA and 10 - deacetylbaccatin III as substrates An effort was made to determine Michaelis Menten kinetic parameters for BAPT with 10 - deacetylbaccatin III (10 - DAB) and (2 R ,3 S ) - PhIS CoA as substrates to product N - debenzoyl - 10 - deacetylpaclitaxel (or N - debenzoyldocetaxel). Under 1 mM substrate concentrations, a V rel of 1.92 × 10 - 4 nmol/min was determined compared with the V rel of 0.018 nmol/min for BAPT with baccatin III using the same preparation of enzyme ( Figure A.3.18 ). Michaelis Menten kinetic constants of BAPT for (2 R ,3 S ) - PhIS CoA and 10 - DAB (at saturatio n) could not be calculated because of a 91.2 - fold reduction in activity with 10 - 187 DAB compared to that for baccatin III. Although BAPT is active with both baccatin III and 10 - DAB, it is possible that active site mutagenesis of BAPT could facilitate more effi cient production of the 10 - deacetylated docetaxel precursor and its analog s. Table 4 . 2 . Kinetic constants of enzymes required for proposed paclitaxel biosynthesis from baccatin III. Substrate 1 K M 1 (µM) Sub strate 2 K M (µM) Substrate 3 K M (µM) k cat (s - 1 ) Ref. Acyl CoA ligases PheAT (2 R ,3 S ) - PhIS (440 ± 62) CoA (208 ± 57) ATP ND 0.015 ± 0.0028 ( 48 ) BadA benzoate (4.4 ± 0.65) CoA (90 - 120) ATP (2 - 3) 28 ± 0.9 (Ch. 2) ( 79 ) Acyltransferases BAPT 2* (2 R ,3 S ) - P hIS CoA (22.4 ± 6.9) - 0.0022 ± 0.00013 This work (3 R ) - - Phe CoA (5.6 ± 1.0) Baccatin III (68 ± 9.6) 0.0583 ± 0.0011 This work NDTNBT Benzoyl CoA (375 ± 67) N - debenzoylpaclitaxel (78 ± 11) ND ( 56 ) Benzoyl CoA (410) N - debenzoyl - - deoxypaclitaxel (450) 1.5 ± 0.3 ( 80 ) *BAPT expressed as MBP - BAPT fusion protein. 1 K M was determined for an enzyme for a given substrate with the additional substrates listed at apparent saturation. 2 - phenylalanine, 3 - hydroxy - phenylpropanoic acid, and 3 - phenylpropanoic acid. Abbreviations: PheAT: p henylalanine CoA ligase from Bacillus subtilis , BadA: benzoate CoA ligase from Rhodopseudomonas palustris , BAPT: baccatin III: 3 - amino - 13 - O - phenylpropanoyl CoA transferase from Taxus cuspidata , NDTNBT: N - debenzoyltaxol - N - benzoyltransferase from Taxus cuspi data . 4.3.4 BAPT activity and substrate assisted catalysis A conservation and homology (ConSurf: consurf.tau.ac.il/) analyses of 150 homologous BAHD family members show complete conservation of the motif, H XXXD, containing the catalytic histidine residue ( 81 - 83 ) . BAPT was unique among the BAHD family members with a G XXXD instead of the conserved H XXXD motif ( Figure 4 . 11 ). This difference is interesting considering the proposed catalytic mechanism for this family of enzym es uses the conserved histidine of the H XXXD motif as a general base to deprotonate the OH or NH 3 + functional group of the acceptor molecule ( Figure 4 . 12 ). The deprotonated acceptor 188 molecule then attacks the electr ophilic center of acyl CoA forming a tetrahedral intermediate, which subsequently collapses to form the acylated acceptor molecule and CoA as a by - product. Clearly, BAPT retains activity without the conserved histidine residue despite reports demonstrating that histidine in the HXXXD motif is catalytically important ( 84, 85 ) . For example, activity was decreased significantly in the BAHD enzymes vinorine synthase and malonyl CoA: anthocyanin 5 - O - glucos ide - - O - malonyltransferase (Ss5MaT1) upon histidine mutation to an inert non - catalytic residue such as alanine ( 84, 85 ) ( Table 4 . 3 ). Loss of the conserved histidine in Ss5MaT1 resulted in a large reduction of the k cat from 8.5 to 0.002 s - 1 , on the same order of magnitude as wild - type BAPT activity with (2 R ,3 S ) - PhIS CoA ( Table 4 . 3 ). Without a BAPT crystal structure to provide context for the non - conserved glycine residue, a homology model was created with RaptorX, a structure - based homology modeling tool ( 86 - 88 ) ( Figure 4 . 13 ). Aligning the BAPT homology model to the hydroxy - cinnamoyl CoA transferase (HCT) acyltransferase from Coffea canephora helps infer a relative position of (3 R ) - - Phe CoA or T. cuspidata T. cuspidat a 2 NPSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGTNVYGSVCDAKGFGQFLQGMAE T. sumatrana KPSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGANVYGSVCDAKGFGQFLQGMAE T. wallichiana* NPSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGTNVYGSVCDAKGFGQFLQGMAE T. x media 1 NPSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGANVYGSTCDAKGFGQFLQGMAE T. x media 2 NPSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGANVYGSTCDAKGFGQFLQGMAE :*************************************:*****.***********.*** * (var. chinensis) Figure 4 . 11 . Multiple sequence alignment of bapt genes from Taxus sp. The G XXX D motif (gray) is shown. Sequences used in the alignment are as follows: T. cuspidata 1 (AAL92459.1), T. cuspidata 2 (AGT51232.1), T. sumatrana (ACN62085.1), T. wallichiana (var. chinensis ) (AGC11862.1), T. x media 1 (AAT73200.1), and T. x media 2 (AFD32416.1). Multiple sequence alignment made with Clustal Omega (EMBL EBI, Cambridge, UK). 189 A. B. Figure 4 . 12 . Proposed mechanisms fo r BAHD acyltransferases and BAPT. A. BAHD acyltransferases with the HXXXD motif utilize the histidine as a base to abstract a proton from the acyl acceptor OH or NH 2 moiety. The acyl acceptor then nucleophilically attacks the acyl donor to form a tetrahedral intermediate (Step I). Subsequent collapse of the intermediate displaces CoASH and forms the acylated accept or (Step II.). B. The proposed substrate - assisted catalytic mechanism for BAPT, which has a non - conserved G XXXD motif. The amino group of (3 R ) - - Phe CoA (R = H) deprotonates the C - 13 hydroxyl group on the acyl acceptor, baccatin III (R 1 = acetyl). The C - 13 oxygen nucleophilically attacks the acyl donor forming a tetrahedral intermediate (Step III) which subsequently collapses, forming N - debenzo ylpaclitaxel and releasing CoA (Step IV). 190 (2 R ,3 S ) - PhIS CoA ( 89 ) . The residues proposed to reside in the active site within 4Å of the glycine residue are Ala40, Ala41, Ala42, Ser44, Val161, Tyr162, Ala165, Cys166, Asn300, and Phe302. There are no obviou s residues that could act as a general base. Polar residues are positioned such that the peptide backbone orients the residue away from the active site, or they are solvent exposed. Although Ser44 is oriented away from the catalytic center of the enzyme, r otation of the sidechain could bring the functional group into the active site ( Figure 4 . 13 ). Tyr162 is modeled in a solvent exposed orientation and Cys166 neighbors the conserved Asp167, pointing away from the act ive site. One limitation of homology modeling of BAHD acyltransferases is the high variability in active site architecture between enzymes, even in structures that bind the same substrate. For instance, malonyl CoA transferases from Nicotiana tabacum (toba cco) (NtMaT1) and red chrysanthemum (Dm3MaT3) both bind malonyl CoA and transfer the malonyl group to an anthocyanin acceptor molecule. However, crystal structures of both enzymes show distinctive active site architectures even though global protein folds are conserved ( 90, 91 ) . An alternative explanation for BAPT activity in the absence of the HXXXD motif is that the free amine of the (3 R ) - - Phe CoA may act as a base in an example of s ubstrate - a ssisted c atalysis (SAC) ( Figure 4 . 12 ) ( 97, 98 ) . In this hypothesis, the (3 R ) - - Phe CoA binds the active site in close proximity to the 3 - amino group. Exampl es of SAC include ( 99, 100 ) , lysozyme ( 101 ) , type II restriction endonucleases ( 102 ) , and poly - ADP - ribose polymerases (PARPs) ( 103 ) . If the amino group of the (3 R ) - - Phe CoA is catalytically important, substrates lacking an amino group shou ld be inactive. Previous work showed BAPT to be inactive with 3 - hydroxy - phenylpropanoyl CoA, - phenylalanyl CoA ( 43, 70 ) . Inactivity with these substrates 191 Table 4 . 3 . Michaelis - Menten k inetic constants from BAHD acyltransferase family me mbers. BAHD acyltransferase Organism Acyl donor 1 Acyl acceptor 1 k cat (s - 1 ) Reference BAPT Taxus cuspidata (2 R ,3 S ) - PhIS CoA (22.4 ± 6.9) Baccatin III 0.0022 ± 0.00013 This work (3 R ) - - Phe CoA (5.6 ± 1.0) Baccatin III (68 ± 9.6) 0.0583 ± 0.0011 This work OsPMT Oryza sativa p - coumaroyl CoA (281) p - coumaryl alcohol (141) 2.6 ( 92 ) PhCFAT Petunia 2 acetyl CoA (30.6) Coniferyl alcohol (56.5) 2.05 ( 93 ) At5MAT Arabidopsis thaliana malonyl CoA (2 - 15) Cy anin (167) 3 0.02 ( 94 ) At5g41040 Arabidopsis thaliana feruloyl CoA (9.7) 16 - OH - palmitic acid (5.1) 0.0013 ( 95 ) EcBAHD7 Erythoxylum coca benzoyl CoA (93) Methylecgonine (369) 9.7 ( 96 ) Ss5MaT1 Salvia splendens malonyl CoA (22) Shisonin (35) 8.7 ( 84 ) Ss5MaT1 H167A malonyl CoA (6.4) Shisonin (5.4) 0.002 1 If available, K M is listed after each substrate (µM). 2 Petunia x hybrid 3 Cyanin is a non - native substrate. suggests the presence and positio n of the 3 - amino group is important for activity. Reduced activity of (2 R ,3 S ) - PhIS CoA suggests the 2 - hydroxyl position may hinder reactivity of the neighboring amine group in the case of SAC, through steric and/or electronic interactions or places the sub strate in a catalytically nonproductive conformation. Previous work in the Walker group (unpublished) showed that a BAPT - Gly163His mutant resulted in no detectable acyl transfer with (3 R ) - Phe CoA, 3 - hydroxyphenylpropanoyl CoA, or phenylpropanoyl CoA to the baccatin III acyl acceptor. These data further support that BAPT evolved to lack the conserved histidine in the HXXXD motif. Without structural support, this hypothesis is difficult to prove, but clearly an alternative base is required in the BAPT active site to facilitate the base - catalyzed mechanism used by this family of enzymes. 192 A. B. Figure 4 . 13 . Homology model of BAPT based on the hydroxycinnamoyl transferase (PDB: 4G0B) from Coffea canephora . - - sheets (red) and loops (purple) are shown. A. An arrow points to the G XXX D (yellow) motif. The active site channel is outlined (circle). Residues proposed to reside within 4 Å of the active site glycine residue include Ala40, Ala41, Ala42 , Ser44, Val161, Tyr162, Ala165, Cys166, Asn300, and Phe302 (not shown). B. Homology model of the BAPT active site aligned with the ligand bound Sorghum hydroxycinnamoyltransferase (HCT) (PDB: 4KEC). HCT products, p - coumarylshikimate and CoA are shown as y ellow sticks and the conserved His162 is shown in magenta. The distance between His162 and the acceptor oxygen of shikimate is 3.3 Å. BAPT residues within 4 Å of the shikimate oxygen are shown. The deviant Gly163 residue and the nearby Ser44 are labeled as well as Arg41, Cys166, Asn300, and Phe302. 193 Figure 4 . 14 . N - debenzoylpaclitaxel biosynthesized from BAPT and PheAT coupled reaction s . Production of N - debenzoylpaclitaxel at two different concentrations of b accatin III at 0.75 mM (gray), 3 mM (dark gray). (n = 2). 4.3.5 Coupled biosynthesis of N - debenzoylpaclitaxel: PheAT and BAPT. As part of developing proof - of - principle for paclitaxel biosynthesis, coupling assays were performed with PheAT, the enzyme capable of (2 R ,3 S ) - phenylisoserine CoA ligase activity. Baccatin III and (2 R ,3 S ) - phenylisoserine were incubated with varied amounts of CoA to determine conditions for maximal production of N - debenzoylpaclitaxel. This was especially important considering the low turn over of PheAT for (2 R ,3 S ) - PhIS CoA and its K M of 208 ± 57 µM for CoA ( Table 4 . 2 ). CoA concentrations were varied from 0.05 to 2 mM and biosynthetic N - debenzoylpaclitaxel was quantified by LC - ESI - MS/MS methods. Ther e appears to be no difference in N - debenzoylpaclitaxel production with low (0.1 mM) or high (2 mM) CoA during PheAT/BAPT coupling with baccatin III (at 0.75 mM or 3 mM) ( Figure 4 . 14 ). However, below 0.1 mM CoA, the production of N - debenzoylpaclitaxel drops significantly, likely because the K M of PheAT for CoA is 208 µM and at concentrations lower than K M , PheAT is not operating at a maximal rate ( V max ). 0 1 2 3 4 5 6 7 8 9 0.05 0.1 0.25 0.5 1 2 N - debenzoylpaclitaxel (nmol) CoA (mM) 194 4.3.6 Biosynthesis of N - 2 - furanoyl - N - debenzoylpaclitaxel: a paclitaxe l analog The four - enzyme (PheAT, BAPT, BadA, and NDTNBT) biosynthesis of paclitaxel was modified due to the presence of 0.1% paclitaxel in our commercial supply of baccatin III. The low 0.1% paclitaxel contamination is easily observed by mass spectrometric analysis, making quantification of biosynthetic paclitaxel difficult. Therefore, to conclusively Figure 4 . 15 . Biosynthesis of N - 2 - furanoyl - N - debenzoylpaclitaxel with PheAT, BAPT, BadA, and NDTNBT. Normalized LC - ESI - MS/MS trace in positive mode for the m / z m / z 509 ion fragmentation. Product peak is at 4.2 min. Background ions are shown for the coupled assay in the absence of NDTNBT. Mass spectrometry performed on a Quattro M icro API LC/MS/MS (Waters, Milford, MA). 195 establish proof of principle for paclitaxel biosynthesis, a biologically active N - 2 - furoyl - N - debenzoylpaclitaxel analog was biosynthesized ( Figure 4 . 15 ) ( 45 ) . The mass o f the analog distinguishes it from the paclitaxel background during LC - ESI/MSMS and 2 - furoyl CoA is 200% as active as benzoyl CoA with NDTNBT and baccatin III ( 55 ) . A clear peak corresponding to m / z m / z 509 appeared at 4.2 min compared to a background control lacking NDTNBT. N - 2 - furoyl - N - debenzoylpaclitaxel was quantified at 1.06 ± 0.08 µM. Levels of N - debenzoylpaclitaxel were 25 ± 9 µm (+NDTNBT) and 39 ± 11 µM ( - NDTNBT). Fragmentation of the [M + H] + = m / z 844 ion was also consistent with taxanes ( Figure A.3.23 ) ( 104, 105 ) . N - 2 - furoyl - N - debenzoylpaclitaxel is one example of an analog that can be made with this system. The combination of PheAT and BAPT yields N - debenzoylpaclitaxel and N - debenzoyl docetaxel precursors ( Figure 4 . 16 ). Substrate specificities of each enzyme define the range of analog s that can be biosynthesized without enzyme engineering for enhanced efficiency or broader substrate specificity. 196 Figure 4 . 16 . Overlapping substrate specificities of PheAT, BAPT, BadA, and NDTNBT. Substrate specificities of each enzyme are represented as circles. Overlapping circles indicate shared substrate spec ificity and the range of compounds that can be biosynthesized by coupling those enzymes together. 197 4.4 Conclusion s (2 R ,3 S ) - PhIS CoA was biosynthesized and isolated on a preparative scale (45.5 mg, 50 µmol) for the first time from a large - scale PheAT CoA liga se - catalyzed reaction. Michaelis - Menten constants of BAPT were calculated for (3 R ) - - Phe CoA, (2 R ,3 S ) - PhIS CoA, and baccatin III. The catalytic efficiency ( k cat / K M ) of BAPT for (2 R ,3 S ) - PhIS CoA ( k cat / K M = 9.82 × 10 - 5 s - 1 µM - 1 ) is 106 - fold lower than for (3 R ) - - Phe CoA ( k cat / K M = 0.0104 s - 1 µM - 1 ) as a result of a decrease in k cat and increases in K M for the former. These results - PhL and NDTNBT, suggesting that enzymes on the natural biosynthetic pathway may prefer to acylate bac catin III with (3 R ) - - Phe CoA over (2 R ,3 S ) - PhIS CoA ( 56, 78 ) . The biologically active, anticancer analog N - 2 - furoyl - N - debenzoylpaclitaxel and the penultimate precursor, N - debenzoylpaclitaxel were produced in coupled enzyme - catalyzed reactions containing bacterial ATP - dependent CoA ligases PheAT and BadA, and Taxus acyltransferases BAPT and NDTNBT. N - Debenzoylpaclitaxel biosynthesized by PheAT and BAPT catalysis is a branchpoint precursor that can be N - acylated, either synthetically or biochemically ( 44 - 47 , 50, 52, 106 ) . PheAT and BAPT coupling also produced the docetaxel precursor N - debenzoyldocetaxel. Development of this system will bypass hurdles met in current efforts to engineer the paclitaxel biosynthetic pathway in heterologous organisms. Baccatin II I and 10 - DAB precursors are readily available at 4 - fold the amount of paclitaxel from yew needles, or Taxus plant cell fermentation bioreactors used to make paclitaxel ( 107, 108 ) paclitaxel biosynthesis system reduces the need to reverse engineer eukaryotic membrane bound P450 hydroxylases to function in bacterial microorganisms. There is minimal flux 198 away from the production of the desired product and no side reactions of the acyltransferases with other taxanes. No more t han four enzymes would need to be optimized for in vivo expression and activity compared with attempting to engineer the entire paclitaxel pathway which requires minimally 19 enzymatic steps. 199 4.5 Future Research Future research with BAPT will include the dev elopment of in vivo assays for the production of paclitaxel. Important factors will include modulation of protein expression, substrate concentrations, and growth effects. Feeding studies in E. coli showed that 10 - DAB can be successfully taken up and acyla ted by 10 - deacetylbaccatin III 10 - O - acetyltransferase (DBAT) in vivo ( 109 ) . Development of a bioengineered in vivo system to produce paclitaxel, analog s, or precursors is likely feasible with feeding of precursors such as baccatin III, 10 - DAB, phenylisoser ine, and acyl acids. Expansion of substrate specificity within the four - enzyme coupled system can be facilitated by rational mutagenesis of enzyme active sites. This has already been demonstrated for BadA with the development of expanded activities and nov el activities from single point mutants. Construction of a PheAT homology model will inform active site mutagenesis to expand the substrate specificity to substituted phenylisoserines in order to make analog s in the C13 - sidechain. Characterization of BAPT substrate specificity with different phenylisoserines will also aid in potential analog production. The synthesis of a series of phenylisoserine derivatives active with PheAT may be coupled to BAPT to test substrate specificities ( 48 ) . Methods to alter th e substrate specificity of BAPT may prove more challenging. The natural mutation of the H XXXD motif to G XXXD in BAPT may affect the orientation of the substrate in the active site, limiting the utility of homology modeling. Another residue may have a catal ytic role, or SAC may occur. Based upon homology modeling, a number of residues are predicted to be in the active site. Mutagenesis of these residues and 200 activity assays will help determine what limits substrate activity. Although BAPT is difficult to puri fy in appreciable yields, crystal screens for the BAPT acyltransferase will b e an important future endeavor. Beyond paclitaxel analog biosynthesis, the combination of plant acyltransferases and microbial acyl CoA ligases may be useful in other bioengineeri ng applications. Acyl CoA ligases are widespread with different substrate specificities. Combinations of acyl CoA ligases and acyltransferases hold potential for generating specific , structurally complex molecular products, as opposed to organic acylation, which may require red unda nt protection reactions, lack regiospecificity, and use toxic organic solvents. 201 APPENDIX 202 APPENDIX Figure A.3.1 . 1 H - NMR of R - N - Boc - 3 - amino - 3 - phenylpropanoic acid . 203 Figure A .3.2 . 1 H - NMR of chemically syn thesized (3 R ) - - phenylalanyl CoA. 204 Figure A .3.3 . HPLC chromatogram of purified (3 R ) - - phenylalanyl CoA. 205 A. B. Figure A .3.4 . Mass spectr a of purified (3 R ) - - phenylalanyl CoA . A. LC - ESI - MS of purified (3 R ) - - phenylalanyl CoA (Exact mass, M = 914.1836). Main peaks correspond to [M - H] - 1 = m / z 913.098, [M - 2H] - 2 = m / z 456.047 . B. LC - ESI/ MSMS of m / z 913. Fragments are labeled. Mass spectrometry was performed on a Xevo G2 - S QTof UPLC/MS/MS (Waters, Milford, MA). 206 Figure A .3.5 . LC - ESI/MSMS chromatogram showing the production of N - debenzoyl - - deoxypaclitaxel . Product was measured by multiple reaction monitoring: m / z m / z 509 by BAPT. Mass spectrometry performed on a Waters Quattro micro API LC/MS/MS (Waters, Milford, MA). Figure A.3. 6 . (Scheme I) Synthesis of (2 R ,3 S ) - phenylisoserinyl C oA . 207 Figure A.3.7 . (Scheme II) (2 R ,3 S ) - PhIS CoA synthesis with 2 - OH prot ection with TBDMS Cl. Figure A.3.8 (Scheme III) ( 2 R ,3 S ) - PhIS CoA synthesis by DCC/ HOBt coupling. 208 Figure A.3.9 ( Scheme IV) ( 2 R ,3 S ) - PhIS CoA synthesis by DCC/NHS coupling. 209 Figure A .3. 10 . 1 H - NMR of ( 2 R, 3 S) - N - Boc - 3 - amino - 3 - phenylisoserine . This compound i s an intermediate in the attempted synthesis of ( 2 R, 3 S) - phenylisoserinyl CoA. 210 Figure A .3. 11 . 1 H - NMR of biosynthetic (2 R ,3 S ) - phenylisoserinyl CoA. 211 Figure A .3. 12 . 13 C - NMR of biosynthetic (2 R ,3 S ) - phenylisoserinyl CoA. 212 Fig ure A .3. 13 . HPLC analysis of purified (2 R ,3 S ) - phenylisoserinyl CoA. 213 A. B. Figure A .3.1 4 . Mass spectra of purified (2 R ,3 S ) - phenylalanyl CoA . A. LC - ESI - MS of purified (2 R ,3 S ) - Phenylalanyl CoA (Exact mass, M = 930.1785). Main peaks correspond to [M - H] - 1 = m / z 929.094, [M - 2H] - 2 = m /z 464.044 . B. LC - ESI/MSMS of m/z 929. Fragments are labeled. Mass spectrometry was performed on a Xevo G2 - S QTof UPLC/MS/MS (Waters, Milford, MA). 214 Figure A .3.1 5 . Michaelis - Menten kinetic plot of biosynthetic N - debenzoylpaclitaxel produced by MBP - BAPT . Substrates were (2 R ,3 S ) - phenylisoserinyl CoA (15 µM to 3 mM) and baccatin III (1 mM). V max = 0.003 ± 1.7 x 10 - 4 nmol/min, K M = 22.35 ± 6.97 µM , k cat = 0.13 ± 0.0076 min - 1 . ( n = 3, n = 2 for 2000 and 3000 µM (2 R ,3 S ) - phenylisoserinyl CoA ). Figure A .3.1 6 . Michaelis - Menten kinetic plot of biosynthetic N - debenzoyl - - deoxypaclitaxel produced by MBP - BAPT . Substrates were (3 R ) - - phenylalanyl CoA (12.5 µM to 1 mM) and baccati n III (1 mM). V max = 0.086 ± 0.0015 nmol/min, K M = 5.6 ± 1.02 µM , k cat = 3.59 ± 0.063 min - 1 . ( n = 3). 215 Figure A .3.1 7 . Michaelis - Menten kinetic plot of biosynthetic N - debenzoyl - - deoxypaclitaxel produced by MBP - BAPT . Substra tes were baccatin III (12.5 µM to 1 mM) and (3 R ) - - phenylalanyl CoA (1 mM). V max = 0.082 ± 0.0026 nmol/min, K M = 68 ± 9.6 µM , k cat = 3.45 ± 0.11 min - 1 . ( n = 3). 216 A. B. Figure A .3.1 8 . BAPT activity with 10 - DAB and (2 R ,3 S ) - PhIS CoA. A. N - debenzoyl - 10 - deacetylpaclitaxel ( m / z m / z 509) and the internal standard docetaxel (1 µM, m / z m / z 509) are shown in black and red respectively. B. Timecourse of BAPT activity. V rel is calculated at 1.9 × 10 - 4 nmol/min with ~57 µg/mL MBP - BAPT (200 µL assay). Mass spectrometry performed on a Waters Quattro micro API LC/MS/MS (Waters, Milford, MA). 217 Figure A .3.1 9 . Purified recombinant PheAT . Purified PheAT is 69.6 kDa. Ladder i s PageRuler Prestained Ladder (Thermo Fisher, Waltham, MA) 218 A. B. C. Figure A .3. 20 . Production of N - debenzoyl - - deoxypaclitaxel by BAPT. A. Structure of N - debenzoyl - - deoxypaclitaxel. B. Total ion chromatogr am of BAPT assay. Enzyme products are at 2.3 and 3.0 min respectively. C. Fragment ions of minor peak (2.3 min) of the N - debenzoyl - - deoxypaclitaxel ion [M +H] + = m / z 734. D. Fragment ions of major peak (3.0 min) of the N - debenzoyl - - deoxypaclitaxel ion [M +H] + = m / z 734. Mass spectrometry performed on a Waters Quattro micro API LC/MS/MS (Waters, Milford, MA). 219 Figure A.3.20 ( cont d ) . D. 220 A. B. C. Figure A .3.21 . Production of N - debenzoylpaclitaxel by BAPT. A. Structure of N - debenzoylpaclitaxel. B. Chromatogram of N - debenzoylpaclitaxel ion [M +H] + = m / z 750. C. Fragment ions of m / z 750. Mass spectrometry performed on a Waters Quattro micro API LC/MS/MS (Waters, Milford, MA). 221 Figure A .3.22 . BAPT and PheAT coupled assay production of N - debenzoylpaclitaxel. Chromatogram of N - debenzoylpaclitaxel ([M+H] + = m / z 750) (black) and the internal standard docetaxel ([M + H] + = m / z 808) (red). Mass spectrometry performed on a Waters Quattr o micro API LC/MS/MS (Waters, Milford, MA). 222 A. B. C. Figure A.3. 23 . 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