‘ . i . .3 .2, I! ,u.‘ 3.... 41.3”? .“Kvytuuwm..: .. . o in; v .. . n . A . . . , . DD. .p , .5 Emma t .ilucl. Ar. 1.. .... . ”muff. ”JFRLMHW .,<&m¢ufiwfi5fi um.....\.flwmfl.w..n. u at. ... i ‘ can ix. .V {(Juuiu . , Jung..— . 1:... Irruar?: L : in... u . . x 5-..... .a .12. . .2233 yuan .. _ 1": lI32A .i... 1 Ian)”: 3 .. .412. I f.!...~0!y..h.:s MC .. «Ms... 2...; 9: .§ f?! 45,323... . 211‘ unit win 1 .11).. 2:: itfiiimk 33:53.... ‘ . 2‘s, .7. . an. , .5. . .3154; .v'71.9u. i .1 V V Quad... 28‘!;(3$n , 1...? .35.. :fifi‘kxww i... f a. .. . i .. a :1 A 33...». .. 33%.».ISIP .. ! ; 3% 56.1% .75. . . > JR I 1.2:...R 3r. _ 3i :1- 4. .: I... 1.... ,.. I}. Atrrn {it I . 21.3%.... I’VIII» '11,! n. . 5M .«éjfixv: h in. a“ A K . .E....:, . l‘. I t x. . 3;... .: .muiat."....hflr..fiu .3: sugar. .... , I! I I THESIS V This is to certify that the dissertation entitled STRUCTURE AND FUNCTION RELATIONSHIPS OF DIHYDRONEOPTERIN ALDOLASES FROM ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS presented by Yi Wang has been accepted towards fulfillment of the requirements for the Ph.D degree in Biochemistry & Molecular Biology éMi 0 7% Major Pro essor’s Signature 5/! 0 /Oé f ' ' Date MSU is an Affinnative Action/Equal Opportunity Institution ~ ~ LIBRARY I Michigan State University _ u.-.--—«-.----o- -a--o---—u—a--a-v-u-I- PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE V JUN 23 {,09' 2/05 p:/C|RC/DaIeDue.indd-p.1 STRUCTURE AND FUNCTION RELATIONSHIPS OF DIHYDRONEOPTERIN ALDOLASES FROM ESCHERICHIA C 0L1 AND S T APH YLOC 0C C US A URE US By Yi Wang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry and Molecular Biology 2006 ABSTRACT STRUCTURE AND FUNCTION RELATIONSHIPS OF DIHYDRONEOPTERIN ALDOLASES FROM ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS By Yi Wang Dihydroneopterin aldolase (DHNA) catalyzes the cleavage of 7,8-dihydro-D- neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). NMR analysis of the reaction products in a D20 solvent suggests that the epimerization reaction follows the same intermediate as the aldol reaction. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism has been determined for DHNA from Staphylococcus aureus (SaDHNA) and DHNA from Escherichia coli (EcDHNA). The results show that they have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them. EcDHNA is different from SaDHNA biochemically in several aspects. EcDHNA has much higher affinities for the substrate, products, and inhibitors measured in this work. EcDHNA has a much higher epimerase activity than SaDHNA. The rate- limiting step is product release for EcDHNA but is the chemical step for SaDHNA. EcDHNA has significantly higher rate constants for the chemical steps than SaDHNA. The functional role of a conserved tyrosine residue at the active site of DHNA has been investigated by site-directed mutagenesis. Comprehensive analysis of the reactions catalyzed by Y54F-SaDHNA and Y53F-EcDHNA showed that the major reaction product is dihydroxanthopterin (DHXP) rather than HP. DHXP is generated via the same enol intermediate as in the wild-type enzyme-catalyzed reaction. The mutants are impaired in the protonation of the enol intermediate to form HP. In addition to the normal products and DHXP, formic acid is also formed in the reaction. In addition to DHNP, molecular oxygen is also consumed in the reaction. The ligand-binding properties of the mutants are perturbed to a small extent. The results showed that the mutant enzymes are oxygenases, and the conserved tyrosine residue plays only a minor role in the physical steps of the enzymatic reaction and the formation of the enol reaction intermediate but a critical role in the protonation of the enol intermediate to form HP. The functional roles of the conserved glutamate and lysine residues at the active site, E22, E74, and K100 in SaDHNA, E21, E73, and K98 in EcDHNA, have been investigated by site-directed mutagenesis in this work. The results showed that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor if any. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis. Copyright by YI WANG 2006 To my parents ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my graduate advisor, Dr. Honggao Yan, for his constant guidance and support. I have benefited greatly from his wealth of knowledge and experience. I am grateful to my graduate committee members, Dr. Robert P. Hausinger for fruitful discussions on mechanistic issues about DHNA, Dr. J. Gregory Zeikus, Dr. Kathleen A. Gallo and Dr. James H. Geiger, for their discussion and suggestions. I thank Dr. Yue Li for her assistance in the site-directed mutagenesis experiments, and Yan Wu for binding and stopped-flow analysis of EcDHNA. Thanks also to Dr. Guangyu Li, Dr. Jaroslaw Blaszczyk and Dr. Krzsztof F elczak, Lishan Yao, Zhenwei Lu, and Jifeng Wang. 1 am also deeply thankful to Dr. Gavin Reid, Gwynyth Anne Scherperel, Dr. Kade D. Roberts and Katherine Rank for MS analysis, Dr. Denise Mills for assistance in the oxygen consumption assay, Mr. James B. McKinlay for assistance in the HPLC identification of formic acid, Dr. A. Daniel Jones for collecting GC-MS data of glycolaldehyde, Mr. Joseph Leykam for expert assistance in the HPLC analysis, Dr. Xinhua Ji and his coworkers for determinations of crystal structures of DHNAs. I am also indebted to Dr. Aizhou Liu, Dr. Daniel Holmes and Dr. Kermit Johnson for training on the operation of NMR, Dr. Zachary F. Burton for reading of the manuscript, Dr. Kaillathe Padmanabhan for solving computer problems. I would also like to thank my friends for their encouragement and grateful help. And finally, I thank my parents and other family members for their support. vi TABEL OF CONTENTS LIST OF TABLES ........................................................................... ix LIST OF FIGURES ......................................................................... x ABBREVIATIONS .......................................................................... xiii CHAPTER 1: INTRODUCTION F olate biosynthesis pathway ................................................................. 1 Biomedical significance of DHNA ......................................................... 4 Uniqueness of DHNA ........................................................................ 9 DHNA is a bifunctional enzyme DHNA is a novel type of aldolases Mechanism studies of DHNA ............................................................... l9 Structures of DHNAs ......................................................................... 20 Active site residues of SaDHNA ............................................................. 25 References ...................................................................................... 27 CHAPTER 2: NMR, EQUILIBRIUM AND TRANSIENT KINETIC STUDIES OF THE S TAPH YLOCOSS US A URE US AND ESCHERICHIA COLI ENZYMES Abstract ......................................................................................... 3 I Introduction .................................................................................... 33 Experimental Procedures ..................................................................... 38 Results .......................................................................................... 43 Discussion ...................................................................................... 63 References ...................................................................................... 69 CHAPTER 3: A POINT MUTATION CONVERTS THE ENZYME TO AN OXYGENASE Abstract ......................................................................................... 72 Introduction .................................................................................... 73 Experimental Procedures ..................................................................... 74 Results .......................................................................................... 7 8 Discussion ...................................................................................... 109 References ...................................................................................... l 18 CHAPTER 4: FUNCTIONAL ROLES OF THE CONSERVED ACTIVE SITE GLUTAMATE AND LYSINE RESIDUES Abstract ........................................................................................ 120 vii Introduction .................................................................................... 12 1 Experimental Procedures ..................................................................... 123 Results .......................................................................................... 12 8 Discussion ...................................................................................... 141 References ...................................................................................... 146 viii LIST OF TABLES CHAPTER 2 Table 2.1. Dissociation Constants of SaDHNA and EcDHNA Measured by Equilibrium Binding Experiments ........................................ 53 Table 2.2. Association and Dissociation Rate Constants of SaDHNA and EcDHNA Measured by Stopped-flow Experiments .................... 57 CHAPTER 3 Table 3.1. Binding Constants of SaDHNA, EcDHNA and Y—>F Mutants ...... 84 Table 3.2. Chemical Shifts of Selected Protons of Compounds Related to the Reactions Catalyzed by the Wild-Type and Mutant DHNAs ......... 88 CHAPTER 4 Table 4.1. The Forward and Reverse Primers for the PCR-Based Mutagenesis Experiments .................................................................. I 25 Table 4.2. Binding Constants of SaDHNA and Site-Directed Mutants ........... 136 Table 4.3. Binding Constants of EcDHNA and Site-Directed Mutants ........... 137 Table 4.4. Steady State Kinetic Constants of SaDHNA and Site-Directed Mutants ........................................................................ 139 Table 4.5. Steady State Kinetic Constants of EcDHNA and Site-Directed Mutants ........................................................................ 140 ix CHAPTER 1 Figure 1.1. Figure 1.2. Figure 1.3. Figure 1.4. Figure 1.5. Figure 1.6. Figure 1.7. CHAPTER 2 Figure 2.1. Figure 2.2. LIST OF FIGURES F olate biosynthesis pathway ............................................... 3 Amino acid sequence assignment of l l DHNAs ........................ 7 The aldolase/epimerase reactions catalyzed by DHNA ................ l2 Mechanisms of two types of aldolases .................................... 15 Proposed mechanism of reactions catalyzed by DHNA ............... 18 The crystal structure of the complex of SaDHNA and product HP. A) Top view, B) Side view ................................................ 22 The potential important residues around the product HP at the active site of SaDHNA ............................................................. 24 The proposed catalytic mechanism for the DHNA -catalyzed reactions ......................................................... 36 Amino acid sequence alignment of DHNAs ............................. 46 Figure 2.3. The SaDHNA-catalyzed reactions in D20 monitored by NMR ....... 49 Figure 2.4. Binding of NP to SaDHNA at equilibrium .............................. 52 Figure 2.5. Stopped-flow analysis of the binding of HPO to EcDHNA ............ 56 Figure 2.6. Quench-flow analysis of the SaDHNA-catalyzed reaction... . .. . ......60 Figure 2.7. Quench-flow analysis of the EcDHNA-catalyzed reaction. . . . . . . . . ....62 Figure 2.8. Summary of the kinetic constants for the SaDHNA-catalyzed (top panel) and EcDHNA-catalyzed (lower panel) reactions... .. ....65 CHAPTER 3 Figure 3.1. Binding of HPO to EcY53F at equilibrium ............................... 81 Figure 3.2. Stopped-flow analysis of the binding of MP to EcY53F ............... 83 Figure 3.3. NMR analysis of the reactions catalyzed by SaDHNA (A) and SaY54F (B) .................................................................. 86 Figure 3.4. BS] MS analysis of the reaction mixtures generated by SaDHNA (A) and SaY54 (B) ......................................................... 90 Figure 3.5. Multistage tandem mass spectrometry identification of the m/z 196 product from the reaction of DHNP with SaDHNA .................... 92 xi Figure 3.6. Multistage tandem mass spectrometry identification of the m/z 182 product from the reaction of DHNP with SaY54F ..................... 95 Figure 3.7. MS spectra of the derivatives of GA Standard (A) and the DHNP reaction mixture generated SaY54F (B) ................................. 98 Figure 3.8. Multistage tandem mass spectrometry identification of the substrate DHNP ........................................................................ 100 Figure 3.9. Multistage tandem mass spectrometry identification of the m/z 194 products from the DHNP reaction catalyzed by SaY54F .............. 102 Figure 3.10. Identification of formic acid by HPLC ................................... 105 Figure 3.11. Time courses of DHNP reactions catalyzed by SaDHNA (A) and SaY54F (B) .................................................................. 108 Figure 3.12. Oxygen consumption by the reactions catalyzed by SaDHNA and SaY54F ....................................................................... 111 Figure 3.13. The proposed chemical mechanism for the generation of DHXP and F DHP ......................................................................... 117 CHAPTER 4 Figure 4.1. Binding of MP to SaE22A at equilibrium ............................... 130 Figure 4.2. Binding of HPO to EcK98A at equilibrium .............................. 132 Figure 4.3. Stopped-flow analysis of the binding of HPO to SaE22A ............. 135 xii CID EcDHNA EcE21A ECE73A EcK98A DEAE DHF R DHMP DHNA DHNP DHNTP DHPS DHXP DSS DTT EcY53F EDTA ESI FDHP FID ABBREVIATIONS collision-induced dissociation dihydroneopterin aldolase from Escherichia coli DHNA with E21 replaced with alanine EcDHNA with E73 replaced with alanine EcDHNA with K98 replaced with alanine diethylaminoethyl dihydrofolate reductase 7, 8-dihydromonapterin dihydroneopterin aldolase 7, 8-dihydroneopterin 7,8-dihydroneopterin triphosphate pyrophosphohydrolase dihydropteroate synthase dihydroxanthopterin 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt dithiothreitol EcDHNA with Y53 replaced with a phenylalanine residue ethylenediaminetetraacetic acid electrospray ionization 6-formyl-7,8-dihydropterin free induction delay xiii FPGS GA GC-MS GTP GTPCH HEPES HiDHNA HMDP HPO HPLC HPPK IPTG LC-MS Ni-NTA NMR NP MP MS MtDHNA MTBSTF A Kd kx pKa folylpolyglutamate synthetase glycolaldehyde gas column with mass spectrometry guanosine triphosphate GTP cyclohydrolase 4-(2-hydroxyethyl)piperazine- l -ethanesulfonic acid dihydroneopterin aldolase from Haemophilus influenzae 6-hydroxymethyl-7,8-hydroxymethyl-pterin 6-hydroxymethyl-pterin high performance liquid chromatography hydroxymethyl-7,8-dihydropterin pyrophosphokinase isopropyl B-D-thiogalactopyranoside liquid column with mass spectrometry nickel-nitrilotriacetic acid nuclear magnetic resonance neopterin monapterin mass spectrometry dihydroneopterin aldolase from Mycobacterium tuberculosis N-(t-butyldimethy1silyl)-N-methyltn'fluoroacetamide dissociation constant rate constants ionization constant xiv SaDHNA SaE22A SaE74A SaK 100A SaKlOOQ SaY54F THF Tris TC EP dihydroneopterin aldolase from Staphylococcus aureus SaDHNA with E22 replaced with alanine SaDHNA with E74 replaced with alanine SaDHNA with K100 replaced with alanine SaDHNA with K100 replaced with glutamine SaDHNA with Y54 replaced with a phenylalanine residue tetrahydrofolate tris(hydroxymethyl).aminomethane tris(2-carboxyethy1)phosphine XV CHAPTER 1: INTRODUCTION F OLATE BIOSYNTHESIS PATHWAY F olate is essential to life. The entire folate synthesis pathway is shown in Figure 1.1. Plants and bacteria can synthesize folate, because they have all of the enzymes in the de novo synthesis pathway (1). Unlike plants and bacteria, humans and other vertebrates are unable to synthesize folate de novo (1). Instead, folic acid is an essential vitamin and humans have to obtain folate from their diet with an active transport system (1). This active transport system does not exist in bacteria. If the de novo folate synthesis pathway is shut down, bacteria can not survive. Therefore, the folate biosynthesis pathway is one of principal targets for the development of antimicrobial agents. The first step in folate biosynthesis is the conversion of GTP into dihydroneopterin triphosphate, which is catalyzed by GTP cyclohydrolase (GTPCH). The triphosphate moiety of dihydroneopterin triphosphate is removed by 7,8- dihydroneopterin triphosphate pyrophosphohydrolase (DHNTP) (2), and the resulting DHNP is converted to 6-hydroxymethy1-7,8-dihydropterin (HP) by dihydroneopterin aldolase (DHNA). The enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to HP. The resulting product is converted to 7,8-dihydropteroate by replacing the pyrophosphate moiety with 4- aminobenzoate, a reaction that is catalyzed by dihydropteroate synthase (DHPS). 7,8- Dihydropteroate is converted to 7,8-dihydrofolate by folylpolyglutamate synthetase (FPGS). Figure 1.1: Folate biosynthesis pathway. GTP, guanosine triphosphate. GTPCH, GTP cyclohydrolase. DHNTP, 7,8-dihydroneopterin triphosphate pyrophosphohydrolase. DHNA, dihydroneopterin aldolase. DHPS, dihydropteroate synthase. HPPK, hydroxymethyl-7,8-dihydropterin pyrophosphokinase. FPGS, folylpolyglutamate synthetase. DHF R, dihydrofolate reductase. Hco, «to HN N j “N HzN’k‘N I N\> GTPCH ’ HzNL‘NI IZW DHNTP H —’2NLN' “SJ/YO“ GTP l :0: I 7,8-dihydroneopterin triphosphate OH 0H "“0 DHNPH DHNA CH, CH HijLfi; jCH, ,NH 000- mil; j,CH,‘_LZ ,ouAMP ATPHNJI; j,CH, ,OH DHPS H NL N HPPK H ,J\N 7.8-dihydro:teroate PPi H,N O C O - 6- -hydroxymethyl- ”7,8 -dihydropterin HP pyrophosphate Glutamate FPGS *ATP . NADP’ ADP +Pi NADPH+H HN 9 L I NTCHZNHQiu-W \ H,N N H H NHOiN $32 H HN C 2 H ?H 9 2 DHFR J§ 9 2 N 9H. M N N W: H 7,8-dihydrofolate C 02- H tetrahydrofolate CO,- 7,8-Dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase (DHF R) using NADPH (3, 4). The first class of antibiotics in clinical use for the treatment of infectious diseases was the sulfonamides, which target DHPS (5). Antibiotics such as trimethoprim have also been developed against DHF R (6). However, the folate pathway is under-explored as a target for developing antimicrobial agents. Both sulfonamides and trimethoprim are still in clinical use, but as for other antibiotics, their clinical use has been severely limited by the development of resistance (7). Antibiotic resistance naturally develops via natural selection through random mutation and plasmid exchange between bacteria of the same species. The mechanisms of resistance and of its spread among pathogenic bacteria show a remarkable evolutionary adaptation to the presence of trimethoprim and sulfonamide. This is reflected in the chromosomal pattern of changes in the structures and mechanisms of regulation of dhps and dhfr genes coding for the target enzymes DHPS and DHFR (7). The rapid development of microbial resistance against current antibiotic drugs requires renewed effort in developing new antimicrobial agents (8). Staphylococcus aureus (S. aureus) has become resistant to many commonly used antibiotics. S. aureus frequently lives on the skin or in the nose of a healthy person. S. aureus can cause illnesses ranging from minor skin infections and abscesses, to life- threatening diseases such as pneumonia, meningitis, endocarditis and septicemia (9). Because of its importance in serious infections, I chose S. aureus as the target organism. BIOMEDICAL SIGNIFICANCE OF DHNA Among the most promising strategies for the development of new antibacterial therapeutics is the targeting of proteins essential for bacterial growth but lacking mammalian counterparts (10). Of the enzymes in the folate pathway, DHNA, HPPK and DHPS are absent in human. Therefore, it is worthwhile to explore other folate biosynthesis enzymes such as DHNA besides DHPS and DHFR enzymes in the folate biosynthesis pathway. DHNA is a particularly attractive target for drug development also because the substrate has no phosphoryl group (11). Several other enzymes such as GTPCH and HPPK in the folate biosynthesis pathway use phosphorylated substrates (e.g. GTP and 6-hydroxymethylpterin pyrophosphate). Nonspecific inhibition of enzymes using substrates with phosphoryl groups is a major problem in developing inhibitors with high specificity (11). Inhibitors of one group of kinases might also inhibit other kinases and phosphatases, and bring global effects on signal transduction pathways. DHNA exists widely in the bacterial kingdom. Generally, there are two types of bacteria: Gram-positive and Gram-negative. S. aureus is an important pathogenic Gram- positive bacterium, and Escherichia coli (E. coli) is a representative of the Gram—negative bacteria. Figure 1.2 shows the multiple sequence alignment of eleven DHNAs from different bacteria. The top five DHNAs are enzymes from Gram-positive bacteria including SaDHNA, and the bottom six DHNAs are enzymes from Gram-negative bacteria including EcDHNA. DHNAs from Gram-positive or Gram-negative bacteria share much similarity with DHNAs in their own group, but fewer similarities are observed between enzymes from the two classes. The identities between enzymes from Gram-positive bacteria range from 39% to 45% and those between Gram-negative bacteria are 49% to 91%, but the identities between Gram-positive and Gram-negative Figure 1.2: Amino acid sequence alignment of 11 bacteria DHNAs. From top to bottom, the 11 DHNAs are from Staphylococcus aureus (SA), Bacillus subtilis (BS), Streptococcus pyogenes (SP), Listeria innocua (LI), Streptomyces coelicolor (SC), Escherichia coli (EC), Salmonella typhi (ST), Yersinia pestis (YP), Vibrio cholerae (VC), Haemophilus influenzae (HI), and Pseudomonas aeruginosa (PA). The First five DHNAs are from Gram-positive bacteria, and the next six DHNAs are from Gram-negative bacteria. The highly conserved residues among all Gram-positive and Gram-negative bacteria are shaded in black. Residues that are characteristics of the Gram-positive or Gram-negative bacteria are shaded in gray. The amino acid sequences are quite different between the enzyme from Gram-positive and Gram-negative bacteria. Many differences are at or near the active site of the enzyme. SA BS SP LI SC ST YP HI PA SA BS LI SC ST YP VC HI PA EQLFNEFPP ------ SEVL QVCLKHE ELLLARF-NS DLLLSRF- ELLLRRF-NS ELIMTRF-A ———< DLLESRY— CLK--- EVLMGER-GI 35 34 34 34 34 34 46 34 121 120 119 124 119 122 120 119 129 118 117 bacterial enzymes are less than 30%. SaDHNA and EcDHNA share only about 23% identity. Many differences between the enzymes from the two classes of bacteria might be at or near their active sites. The positions of their active sites are indicated by the crystal structures of SaDHNA. Lower sequence similarities indicate that Gram-positive and Gram-negative enzymes might have different characteristics. It may therefore be possible to design a “narrow spectrum” antibiotic drug for DHNA. The major modification sites on the pterin ring for inhibitor design of DHNA are C6 and C7. Potent inhibitors of DHNA have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization (10). Several lead compounds with ICso values of about 1 uM against DHNA were identified among a 10,000 random compound library. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar ICso values (10). The potential problem with the crystallographic screening is that the binding pocket is fixed in one conformation in the crystallized form used for soaking-in of the ligand of interest. More inhibitors can be found when different conformations of DHNA are available in solution. A significant improvement of inhibition can be achieved by structure-based design and through additional cycles of screening (10) UNIQUENESS OF DHNA l) DHNA is a bifunctional enzyme DHNA catalyzes both an aldolase and epimerase reaction as shown in Figure 1.3. As an aldolase, it converts the natural substrate DHNP to HP and glycolaldehyde (GA). As an epimerase, DHNA converts DHNP to 7,8-dihydromonapterin (DHMP), a stereoisomer of DHNP. The steady-state kinetic parameters of EcDHNA and DHNA from Haemophilus influenzae (HiDHNA) from the catalyzed reaction were determined in 1998 (12). DHNP and DHMP both can be used as substrates and their aldolase reaction activities kw, are 127 and 158 umol/mg/h, respectively. Km values for DHNP and DHMP are 64 and 36 M, respectively. HiDHNA has a higher aldolase reaction activity than EcDHNA with DHNP as substrate, but it has a lower aldolase activity than EcDHNA with DHMP as substrate. Its epimerase activity with DHNP and DHMP as substrates is also lower than that of EcDHNA (12). The fact that DHNA has an epimerase activity is not totally surprising because the enzyme is homologous to dihydroneopterin triphosphate epimerase, which catalyzes the stereochemical conversion of dihydroneopterin triphosphate and dihydromonapterin triphosphate. The sequence identity between DHNA and dihydroneopterin triphosphate epimerases from E. coli is 30%, higher than the 23% identity between SaDHNA and EcDHNA. Furthermore, the two enzymes have a similar folding topology and quaternary structure (13). The epimerase also has a low level of aldolase activity. Because these enzymes each have two activities, it is proposed that the aldolase and epimerase reactions share a common reaction intermediate. A retro-aldol cleavage of the C-C bond between C l ’ and C2’ is proposed to be a crucial step in both reactions (12). For the epimerase reaction, the intermediate species stay long enough in the active site of the enzyme so that one of the cleaved products can rotate and reattach to form a new stereoisomer. For the aldolase reaction, two products may be released quickly from the active site of the enzyme. The overall products of these enzymatic reactions may be determined by the relative rates of these processes. It is reasonable to predict that aldolase could become an epimerase if the reaction intermediates are trapped at the active site to allow them to rotate and re-form the C-C bond. Another pair of enzymes shares relationships with DHNA and dihydroneopterin triphosphate epimerase, namely L-fuculose-l-phosphate aldolase and ribulose-S— phosphate-epimerase. L-Fuculose-l-phosphate aldolase catalyzes the reversible cleavage of L-fuculose-l-phosphate to dihydroxyacetone phosphate and L-lactaldehyde (14). Ribulose-S-Phosphate-epimerase catalyzes the interconversion of L-ribulose-S-phosphate and D-xylulose-S-phosphate (15). The two enzymes both have a divalent cation at their active sites (16). 10 Figure 1.3: The aldolase/epimerase reactions catalyzed by DHNA. ll N O HN \ OH R HzN N N AldOIaSe H Reaction HP GA 9H N ' 2, HN I W0” H NkN N 7 0H DHNP N\ Epimerase HN I 5 OH Reaction /i\\ OH HZN N N H DHMP 12 Moreover, L-ribulose 5-phosphate 4-epimerase uses a retroaldol/aldol mechanism for the epimerization of L-ribulose 5-phosphate. The evidence for the mechanism is solid: (a) No solvent isotope incorporation (2H or 180) could be detected, indicating that any mechanism involving nonstereospecific deprotonation/reprotonation is highly unlikely (15). (b) There were no primary deuterium isotope effects on the protons at either C-3 or C4 (17). (c) '3 C kinetic isotope effects were observed on both the C3 carbon (1.85%) and the C4 carbon (1.5%) of L-ribulose 5-phosphate, which is consistent with a mechanism involving C-C bond cleavage (1 7). ((1) There is a detectable aldolase activity (18). (e) The epimerase shares 26% sequence identity with L-fuculose-l-phosphate aldolase as described earlier (14). When the structures of the epimerase and the aldolase are superimposed, 93% of the or-carbons align with a root-mean square deviation of only 1.5 A, indicating that the two enzymes belong to a superfamily of aldolases/epimerases and have evolved from a common ancestor (19). 2) DHNA is a novel aldolase There are two known classes of aldolases (20, 21). Class I aldolases utilize an active-site lysine to form a Schiff base to activate their substrates, while class II enzymes require a divalent metal ion, usually zinc, for catalysis as shown in Figure 1.4 (22). Type I aldolases include fructose-1,6-bisphosphate aldolase (23), 2-keto-3-deoxy-6- phosphogluconate aldolase (24), and N-acetylneuraminate aldolase (25). Type II enzymes 13 Figure 1.4: Mechanisms of two types of aldolases. 14 ”TM yS His ..H-N/N/ N“‘-Zn2+ ”N O :NH + Type 1 Aldolase (U \ X H R. R O / acceptor R/“VX ”r” Type 2 Aldolase 0) 3 JV. 11 R H R' V acceptor include L-fuculose-l-phosphate aldolase (26), fructose-1,6-bisphosphate aldolase (27, 28) and L-rhamnulose-l-phosphate aldolase (29, 30). DHNA does not form a Schiff base with its substrate as found in type I aldolases nor does it need a metal ion for catalysis as for type II aldolases. Instead, the Schiff base appears to be embedded in the substrate DHNP/DHMP itself as shown in Figure 1.5. Specifically, the protonation of N5 in the substrate helps to stabilize the enol reaction intermediate. Without the imine group at N5 functioning as a Schiff base, the aldolase reaction would not proceed. NP is the oxidized form of substrate DHNP and tetrahydroneopterin is the reduced from of substrate DHNP. They cannot produce a protonated imine group at N5 and neither NP nor tetrahydroneopterin can be used as a substrate (31). Tetrahydroneopterin does not contain the imine required by the reaction and the aromatic nature of neopterin would deactivate the imine through delocalization (32). 16 Figure 1.5: Proposed mechanism of reactions catalyzed by DHNA. l7 Hi | 5‘61' 3'OH HN I 5\61v,3'orr HN N N 7 kN N 7 O. 2 H HZN H8 .... \ / o H , 1;, 1 ”FBI ECO” OH‘ \ + H,N N E o H Intermediate GA 0 1, N O HN \ OH \ J\\ I + H H N N N 7 0H 2 H8 18 MECHANISTIC STUDIES OF DHNA Although the proposed chemical mechanism of the DHNA-catalyzed reaction is reasonable, no experimental evidence has been reported and how the enzyme catalyzes the reaction is largely unknown (32). In the proposed reaction mechanism, C2’-C3’ cleavage occurs with formation of an enol and a proton is transferred to the enol intermediate to form the product HP as shown in Figure 1.5 (33). The mechanism of the protonation transfer was studied by NMR in 2002 (9). In order to determine how a proton is transferred in the step between the enol intermediate and the product HP, the incorporation of deuterium from the solvent D20 into the heterocyclic reaction product was measured by multinuclear NMR spectroscopy by dissolving the enzyme and substrate in D20. The data indicated that the protonation of the intermediate by DHNA occurs preferentially in the pro-S position. Although the data do not answer the question of whether or not the proton donor is an acidic group of the protein or solvent water, it is clear that the protonation of the enol intermediate does not occur by a return of the proton that had been abstracted from the substrate (9). A study using Raman difference spectroscopy of the electronic structure of dihydrobiopterin, the analogue of DHNP, bound to SaDHNA indicates that the pKa of N5 is not significantly increased in the enzyme-bound form (34). Dihydrobiopterin stays in an unprotonated form. This result suggested that N5 of DHNP might not be protonated before the bond cleavage of DHNP during the DHNA-catalyzed reaction (34). 19 STRUCTURES OF DHNAS The study of the structure and function relationship of DHNA is based on the high resolution X-ray crystal structure. DHNA was first identified in E. coli in 1970 (31 ). The genes of both EcDHNA (12) and SaDHNA (32) were cloned and overproduced in E. coli by recombinant DNA methods in 1998. Crystal structure of the binary complex form of SaDHNA and product HP was determined at 1.65 A resolution by Hennig and coworkers (32). SaDHNA is a homooctamer with 121 residues of 13.8 kDa per subunit. The octameric structure is composed of two stacked rings each composed of four subunits. Each subunit contains a “T-fold” structure with one antiparallel [3 sheet and two or helices. For each tetramer, the subunits are placed “head to head” to form a circular “donut”- shaped ring with a big hole in the middle. The overall quaternary structure is similar in appearance to two stacked “donuts”. The top-down view and side view of the protein in complex with the product HP are shown in Figure 1.6A and 1.68, respectively. The crystal structure of active site residues is shown in Figure 1.7. Figures 1.6 and 1.7 are from the Protein Data Bank structure of 2dhn (32). In 2000, the sequential resonance assignment of the HO kDa SaDHNA was determined by NMR. The protein was labeled with 2H, '3C, and ”N uniformly to increase the sensitivity of the measurements. The complete sequence assignment of the octamer was obtained, and the regular secondary structures in the solution conformation were found to coincide nearly identically with those in the crystal structure (35). 20 Figure 1.6: The crystal structure of the complex of SaDHNA and product HP. A) Top view, B) Side view. The small molecules located at the interface between two adjacent subunits are product HP at active sites. 21 W5.“ ’ '/ f ‘3’ cfx V .D V it» 4 P. ) B A) 22 Figure 1.7: The potentially important residues around the product HP at the active site of SaDHNA. The residues shown are E22, Y54, E74, and K100. A water molecule is found close to N5 of HP. The dotted lines represent hydrogen bonds. 23 24 The crystal structure of DHNA from Arabidopsis thaliana (AtDHNA) has been determined at 2.2 A resolution (33). The enzyme forms a D4-symmetric homooctamer, structurally similar to SaDHNA. It has 126 residues in each monomer, each of 14.1 kDa. The enzyme is bound with guanine. The spatial arrangements of active site residues of AtDHNA are superimposable to those of SaDHNA (33). The 1.6 A X-ray crystal structure of DHNA from Mycobacterium tuberculosis (MtDHNA) complexed with the product HP reveals an octameric assembly similar to SaDHNA (28). However, the 2.5 A crystal structure of unliganded MtDHNA reveals a novel tetrameric oligomerization state, with only partially formed active sites. Unlike SaDHNA and EcDHNA, MtDHNA displays cooperativity in substrate binding, which is proposed to regulate the cellular concentration of DHNP so that it may fimction not only as a precursor for folate biosynthesis but also as an antioxidant for the survival of the organism against host defense (36). ACTIVE SITE RESIDUES OF SADHNA The octameric protein contains eight active sites with each active site located at the interface of two adjacent subunits (Figures 1.6 and 1.7) (32). Each active site contains four conserved residues, E22, E74, and K100 from one subunit and Y54 from the adjacent subunit (SaDHNA numbering, Figure 1.7). HP is stacked with the phenol ring of Y54 and is fixed at the active site by five hydrogen bonds with the protein (including the two hydrogen bonds with main chain amides, not shown). The carboxyl group of E22 forms a hydrogen bond with the hydroxyl group of HP and with the amino group of K] 00. 25 The hydroxyl group of the phenol ring of Y54 forms a hydrogen bond with the hydroxyl group of HP as well as with the amino group of K100. The carboxyl group of E74 forms two hydrogen bonds with HP. In addition to the hydrogen bonds with E22 and Y54, K100 also forms a hydrogen bond with a water molecule, which is in turn hydrogen bonded to N5 of HP. It is noted that there is no residue in SaDHNA that can form hydrogen bond directly with N5 of HP. While the crystal structures of SaDHNA, AtDHNA and MtDHNA have provided a three-dimensional view of the active sites of the enzymes, the functional roles of the active site residues are yet to be established. In order to answer how DHNA catalyzes the aldolase and epimerase reactions, thermodynamic and kinetic analyses of SaDHNA and EcDHNA were carried out first (Chapter 2). Subsequently, site-directed mutagenesis was used to remove the functional groups at the active sites of the enzymes, and changes in the binding and catalytic properties of the enzymes were measured to determine the functional roles of these residues (Chapter 3 and 4). Of particular interest, mutations involving the active site Tyr residue were found to confer unique new chemistry to the enzyme such that oxygenase activity was identified (Chapter 3). 26 REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) Young, D. W. (1986) The biosynthesis of the vitamins thiamin, riboflavin, and folic acid. Nat. Prod. Rep. 3, 395-419. . Klaus, S. M. J ., Wegkamp, A., Sybesma W., Hugenholtz, J., Gregory, J. F. 111, and Hanson A. D. (2005) A Nudix Enzyme Removes Pyrophosphate from Dihydroneopterin Triphosphate in the F olate Synthesis Pathway of Bacteria and Plants. J. Biol. Chem. 280, 5274-5280. Berrningham, A., and Derrick, J. P. (2002) The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery. Bioassays. 24. 637-648. Kompis, I. M., Islam, K., and Then, R. L. (2005) DNA and RNA Synthesis: Antifolates. Chem. Rev. 105. 593-620. Chio, L. C., Bolyard, L. A., Nasr, M., and Queener, S. F. (1996) Identification of a class of sulfonamides highly active against dihydropteroate synthase form Toxoplasma gondii, Pneumocystis carinii, and Mycobacterium avium. Antimicrob. Agents Chemother. 40, 727-733. Garg, S. K., Ghosh, S. S., and Mathur, V. S. (1986) Comparative pharmacokinetic study of four different sulfonamides in combination with trimethoprim in human volunteers. Int. J. Clin. Pharmacol. T her. T oxicol. 24, 23-25. Huovinen, P., Sundstrém, L. Swedberg, G., and Skéild, O. (1995) Trimethoprim and sulfonamide resistance. Antimicrob. Agents Chemother. 39, 279-289. Illarionova, V., Eisenreich, W., Fischer, M., Haussmann, C., Romisch, W., Richter, G., and Bacher, A. (2002) Biosynthesis of tetrahydrofolate - stereochemistry of dihydroneopterin aldolase. J. Biol. Chem. 277, 28841-28847. Todar, K. (2005) Todar's Online Textbook of Bacteriology. University of W isconsin-Madison Department of Bacteriology. Sanders, W. J., Nienaber, V. L., Lerner, C. G., McCall, J. O., Merrick, S. M., Swanson, S. J ., Harlan, J. E., Stoll, V. S., Stamper, G. F., Betz, S. F., Condroski, K. R., Meadows, R. P., Severin, J. M., Walter, K. A., Magdalinos, P., Jakob, C. G., Wagner, R., and Beutel, B. A. (2004) Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization. J. Med. Chem. 47,1709-1718. 27 (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) Zimmerman, M., Tolman, R. L., Morman, H., Graham, D. W., and Rogers, E. F. (1977) Inhibitors of folate biosynthesis .1. Inhibition of dihydroneopterin aldolase by pteridine derivatives. J. Med. Chem. 20, 1213-1215. HauBmann, C., Rohdich, F., Schmidt, E., Bacher, A., and Richter, F. (1998) Biosynthesis of pteridines in Escherichia coli - structural and mechanistic similarity of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase. J. Biol. Chem. 273, 17418-17424. Ploom, T., Haussmann, C., Hof, P., Steinbacher, S., Bacher, A., Richardson, J., and Huber, R. (1999) Crystal structure of 7,8-dihydroneopterin triphosphate epimerase. Structure 7, 509-16. Dreyer, M. K., and Schulz, G. E. (1993) The spatial structure of the class II [- fuculose-l-phosphate aldolase from Escherichia coli. J. Mol. Biol. 231, 549-533. Tanner, M. E., and Kenyon, G. L. (1998) Inversion of stereocenters., Vol. II, Academic Press, San Diego. Samuel, J., Luo, Y., Morgan, P. M., Strynadka, N. C. J., and Tanner, M. E. (2001) Catalysis and binding in l-ribulose-S-phosphate 4-epimerase: A comparison with l-fiiculose-l-phosphate aldolase. Biochemistry 40, 14772-14780. Lee, L. V., Vu, M. V., and Cleland, W. W. (2000) 13C and deuterium isotope effects suggest an aldol cleavage mechanism for l-ribulose-S-phosphate 4- epimerase. Biochemistry 39, 4808-4820. Johnson, A. E., and Tanner, M. E. (1998) Epimerization via carbon-carbon bond cleavage. L-ribulose-S- phosphate 4-epimerase as a masked class II aldolase. Biochemistry 3 7, 5746-5754. Tanner, M. E. (2002) Understanding nature's strategies for enzyme-catalyzed racemization and epimerization. Acc. Chem. Res. 35, 237-46. Horecker, B. L., Tsolas, O., and Lai, C.-Y. (1975) Aldolases, in The enzymes (Boyer, P. D., Ed.) pp 213-258, Academic Press, San Diego. Allen, K. N. (1998) Reactions of enzyme-derived enamines, in Comprehensive biological catalysis (Sinnott, M., Ed.) pp 135-172, Academic Press, San Diego. Silverman, R. B. (2000) The organic chemistry of enzyme-catalyzed reactions pp 455, Academic Press, San Diego. Lorentzen, E., Siebers, B., Hensel, R., and Pohl, E. (2005) Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: Structural analysis of reaction intermediates. Biochemistry 44, 4222-9. 28 (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) Mavridis, I. M., Hatada, M. H., Tulinsky, A., and Lebioda, L. (1982) Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase at 2.8 a resolution. J. Mol. Biol. 162, 419-444. Lawrence, M. C., Barbosa, J. A. R. G., Smith, B. J., Hall, N. E., Pilling, P. A., Ooi, H. C., and Marcuccio, S. M. (1997) Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase. J. Mol. Biol. 266, 381-399. Dreyer, M. K., and Schulz, G. E. (1996) Refined high-resolution structure of the metal-ion dependent l-fuculose-l-phosphate aldolase (class II) from Escherichia coli. Acta Crystallogr., Sect. D 52, 1082-1091. Hall, D. R., Leonard, G. A., Reed, C. D., Watt, C. 1., Berry, A., and Hunter, W. N. (1999) The crystal structure of Escherichia coli class II fructose- 1,6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity. J. Mol. Biol. 287, 383-394. Cooper, S. J ., Leonard, G. A., McSweeney, S. M., Thompson, A. W., Naismith, J. H., Qamar, S., Plater, A., Berry, A., and Hunter, W. N. (1996) The crystal structure of a class II fructose-1,6-bisphosphate aldolaseshows a novel binuclear metal-binding active site embedded in a familiar fold. Structure 4, 1303-1315. Schwartz, N. B., Abram, D., and F eingold, D. S. (1974) L-Rhamnulose 1- phosphate aldolase of Escherichia coli. The role of metal in enzyme structure. Biochemistry 13, 1726-30. Chiu, T. H., and Feingold, D. S. (1969) L-Rharrmulose 1-phosphate aldolase from Escherichia coli. Crystallization and properties. Biochemistry 8, 98-108. Mathis, J. B., and Brown, G. M. (1970) The biosynthesis of folic acid XI. Purification and properties of dihydroneopterin aldolase. J. Biol. Chem. 245, 3015-3025. Hennig, M., D'Arcy, A., Hampele, I. C., Page, M. G. P., Oefiier, C., and Dale, G. E. (1998) Crystal structure and reaction mechanism of 7,8- dihydroneopterin aldolase from Staphylococcus aureus. Nature Struct. Biol. 5, 357-362. Bauer, S., Schott, A. K., Illarionova, V., Bacher, A., Huber, R., and Fischer, M. (2004) Biosynthesis of tetrahydrofolate in plants: Crystal structure of 7,8- dihydroneopterin aldolase from Arabidopsis thaliana reveals a novel adolase class. J. Mol. Biol. 339, 967-79. Deng, H., Callender, R., and Dale, G. E. (2000) A vibrational structure of 7,8- dihydrobiopterin bound to dihydroneopterin aldolase. J. Biol. Chem. 275, 30139- 30143. 29 (35) (36) Salzmann, M., Pervushin, K., Wider, G., Senn, H., and Wiithrich, K. (2000) NMR assignment and secondary structure determination of an octameric 110 kDa protein using TROSY in triple resonance experiments. .1. Am. Chem. Soc. 122, 7543-7548. Goulding, C. W., Apostol, M. 1., Sawaya, M. R., Phillips, M., Parseghian, A., and Eisenberg, D. (2005) Regulation by oligomerization in a mycobacterial folate biosynthetic enzyme. J. Mol. Biol. 349, 61-72. 30 CHAPTER 2: NMR, EQULIBRIUM AND TRANSIENT KINETIC STUDIES OF STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI ENZYMES ABSTRACT Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro- D-neopterin (DHNP) to form 6-hydroxymthyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form, 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as in the aldol reaction or the deprotonation and re-protonation of the 2’-carbon of DHNP has been investigated by NMR analysis of the reaction products in a D20 solvent. No deuteration of the 2’-carbon was observed for the newly formed DHMP, while there was a significant deuteration of the 6-hydroxymethyl carbon of HP. The results strongly suggest that the epimerization reaction uses the same reaction intermediate as the aldol reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) have been determined by equilibrium binding and transient kinetic studies. The DHNA-catalyzed reaction is reversible in contrast to an early observation. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism has been determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them. EcDHNA is different from SaDHNA biochemically in several aspects. (1) EcDHNA has much higher affinities for the 31 substrate, products, and inhibitors measured in this work. (2) EcDHNA has a much higher epimerase activity than SaDHNA. (3) The rate-limiting step in the forward reaction (the formation of HP) is the product release for EcDHNA but is the formation of the reaction intermediate for SaDHNA. (4) The reverse reaction is very slow with EcDHNA but very fast with SaDHNA. 32 INTRODUCTION Infectious diseases are the leading causes of death and the main causes of premature death (0-44 years) (2). Widespread and persistent antibiotic resistance has caused a worldwide health care crisis (3-5). The crisis has been aggravated by the decisions by many major pharmaceutical companies to abandon or curtail their antibacterial programs for business reasons (6-8), and the fact that most new antibiotics are chemical modifications of existing antimicrobial agents (9). These compounds act against old targets and are therefore less effective in dealing with widespread antibiotic resistance. New targets for the development of novel antimicrobial agents are thus urgently needed for combating the antibiotic crisis. Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8- dihydroneopterin (DHNP) to 6-hydroxymethy1-7,8—dihydropterin (HP) in the folate biosynthetic pathway, one of principal targets for developing antimicrobial agents (3). F olate cofactors are essential for life (10). Most microorganisms must synthesize folates de novo. In contrast, mammals cannot synthesize folates because of the lack of three enzymes in the middle of the folate pathway and obtain folates from the diet. DHNA is the first of the three enzymes that are absent in mammals and therefore an attractive target for developing antimicrobial agents (11). DHNA is a unique aldolase in two respects. First, DHNA requires neither the formation of a Schiff’s base between the substrate and enzyme nor metal ions for catalysis (12). Aldolases can be divided into two classes based on their catalytic mechanisms (13, 14). Class I aldolases require the formation of a Schiff’s base between 33 an amino group of the enzyme and the carbonyl of the substrate, whereas class II aldolases require a Zn2+ ion at their active sites for catalysis. The proposed catalytic mechanism for DHNA (12, 15, 16) is similar to that of class I aldolases, but the Schiff’s base is embedded in the substrate (Figure 2.1). Secondly, in addition to the aldolase reaction, DHNA also catalyzes the epimerization at the 2’-carbon of DHNP to generate 7,8-dihydromonapterin (DHMP) (15), but the biological function of the epimerase reaction is not known at present. The aldolase and epimerase reactions are believed to involve a common intermediate as shown in Figure 2.1 (12, 15, 16). Both reactions involve the retroaldol cleavage of the C-C bond between Cl’ and C2’. Epimerization results from the re-formation of the C-C bond after the rotation of glycolaldehyde. The mechanism of the epimerization reaction is very similar to that Catalyzed by L-ribulose-S- phosphate 4-epimerase (17), which also follows aldol chemistry (18), but the two enzymes are different in structure and have no apparent sequence identity. L-ribulose-S- phosphate 4-epimerase has 26% identity with the class II L-fuculose-l-phosphate aldolase and requires a Zn2+ ion for catalysis (17). DHNA is unique because it catalyzes both aldolase and epimerase reactions, whereas L-ribulose-S-phosphate 4-epimerase and L-fuculose-l-phosphate aldolase catalyze only one type of reaction. Interestingly, DHNAs from Gram-positive and Gram-negative bacteria have some unique sequence motifs. Figure 1.2 shows the amino acid sequence alignment of DHNAs from 11 bacteria. The first five enzymes are from Gram-positive bacteria, and the rest are from Gram-negative bacteria. The identities between enzymes from Gram-positive bacteria range from 39% to 45% and those between Gram-negative bacteria are 49-91%, but the identities between Gram-positive and Gram-negative bacterial enzymes are 34 Figure 2.1: The proposed catalytic mechanism for the DHNA-catalyzed reactions. 35 A-H A-H o ) 9H 0 3 QH 3 2| N 1 HN3 23 a; 3. OH HN I \ u , OH \1 I 8 o? )\\ o? H2N N N LH H2N N 11‘. H B DHNP ‘3 DHMP ' A-z’V .3" 0 1'4 OH 0 l-l-l OH Nj/ Nn/ HN l C, // \ T Hi]: I C '1' OH _. + ‘ \ H—B HZNJFN n ”L? O 0” HzN N INI L. OH \ o A—H / HN N\ OH °\\ HzN/KJIP‘f :B + OH N Hp glycolaldehyde (GA) 36 <30%. Many differences between enzymes from Gram-positive and Gram—negative bacteria are at or near their active centers (16). DHNA was first identified in Escherichia coli (EcDHNA) by Mathis and Brown in 1970 (12). There were few studies on DHNA until 1998, when Hennig and coworkers determined the crystal structures of DHNA from Staphylococcus aureus (SaDHNA) and its complex with the product HP (16). In the same year, HauBmann and coworkers demonstrated that the enzyme has both aldolase and epimerase activities and determined the steady-state kinetic parameters for both reactions (15). In 2000, the Witthrich group published the total sequential resonance assignment of the 110 kDa homooctomeric SaDHNA (19), which was a model system for the development of TROSY NMR spectroscopy (20-22). Also in 2000, Deng and coworkers measured the pK, of nitrogen 5 of SaDHNA-bound 7,8-dihydrobiopterin by Raman spectroscopy (23). In 2002, lllarionova and coworkers showed that the protonation of the reaction intermediate prefers the pro-S position (24). Most recently, we studied the dynamic properties of apo SaDHNA and the product complex SaDHNA-HP by molecular dynamics simulations (1). How DHNA catalyzes both aldol and epimerization reactions is however largely unknown at present. We are interested in understanding the catalytic mechanism of DHNA and the biochemical consequences of the significant sequence differences described above. To this end, we performed a comprehensive equilibrium and kinetic study of SaDHNA and EcDHNA, representing DHNAs from Gram-positive and Gram-negative bacteria, respectively. We also addressed the issue of whether the epimerase reaction uses the same reaction intermediate as that of the aldolase reaction or an alternative mechanism 37 via the deprotonation and re-protonation of 2’-carbon. The results showed that the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction and that SaDHNA and EcDHNA have significantly different equilibrium and kinetic constants, which form the basis for elucidating the catalytic mechanism of DHNA and developing antimicrobial agents specifically against Gram-positive or Gram-negative bacteria. EXPERIMENTAL PROCEDURES Materials. 6-Hydroxymethylpterin (HPO), 6-hydroxymethyl-7,8-dihydropterin (HP), 7,8-dihydro-D-neopterin (DHNP), 7,8-dihydro-L-m0napterin (DHMP), D- neopterin (NP), and L-monapterin (MP) were purchased from Schircks Laboratories. Restriction enzymes and T4 ligase were purchased from New England Biolabs. Pfu DNA polymerase and the pET-17b vector were purchased from Strategene and Novagen, respectively. Other chemicals were from Sigma or Aldrich. Cloning. The SaDHNA gene was cloned into the prokaryotic expression vector pET-17b and a home-made derivative (pET17H) by PCR from S. aureus genomic DNA. The pET17H vector was used for the production of a His-tagged SaDHNA. The primers for the PCR reaction were 5’-G GAA TTC CAT ATG CAA GAC ACA ATC TTT CTT AAA G -3’(forward primer with a Nde I site) and 5’- CG GGA TCC TCA TTT ATT CTC CCT CAC TAT TTC-3’ (reverse primer with an BamH I site). The EcDHNA gene was cloned into the prokaryotic expression vector pET-17b by PCR from E. coli genomic DNA. The primers for the PCR reaction were 5’—G GAA TTC CAT ATG GAT ATT 38 GTA TTT ATA GAG CAA C -3’ (forward primer with a Nde I site) and 5’- CG GGA TCC TTA ATT ATT TTC TTT CAG ATT ATT GCC-3’ (reverse primer with an BamH I site). The expression constructs were transformed into the E. coli strain DH5a. The correct coding sequences of the cloned genes were verified by DNA sequencing. The verified SaDHNA expression constructs were transformed into the E. coli strain BL21(DE3)pLysS for over-production of SaDHNA. The verified EcDHNA expression construct was transformed into the E. coli strain BL21 (DE3) for over-production of EcDHNA. Expression and Purification. The non-tagged SaDHNA was purified by ion exchange chromatography on a DEAE-cellulose column and gel filtration on a Bio-Gel A-0.5m gel column. One liter of LB medium containing 100 mg ampicillin and 20 mg chloramphenicol was inoculated with 5 mL of overnight seed culture and incubated at 37 °C with vigorous shaking. The production of the SaDHNA was induced when the OD600 of the culture reached 0.8-1.0. The culture was further incubated for 4 h and harvested by centrifugation. The E. coli cells were re-suspended in 20 mM Tris-HCl, pH 8.0 (buffer A) and lysed by a French press. The lysate was centrifuged for 20 min at ~27,000 g. The supernatant was loaded onto a DEAE-cellulose column equilibrated with buffer A. The column was washed with buffer A until 0mm of the effluent was <0.05 and eluted with a 0-500 mM linear NaCl gradient in buffer A. Fractions containing DHNA were identified by Ongo and SDS-PAGE and concentrated to ~15 mL by an Amicon concentrator using a YM30 membrane. The concentrated protein solution was centrifuged, and the supernatant was applied to a Bio-Gel A-0.5m gel column equilibrated with buffer A containing 150 mM NaCl. The column was developed with the same buffer. Fractions 39 from the gel filtration column were monitored by Ongo and SDS-PAGE. Pure DHNA fractions were pooled and concentrated to 10-20 mL. The concentrated DHNA was dialyzed against 5 mM TrisHCl, pH 8.0, 1yophilized, and stored at -80 °C. EcDHNA was purified essentially the same way except that the E. coli cells that over-produced EcDHNA were from overnight cultures from single colonies without the IPTG induction. The His-tagged SaDHNA was purified by a Ni-NTA column and a Bio-Gel A-0.5m gel column. The cells were harvested and lysed as described above except that buffer was replaced with 50 mM sodium phosphate, 300 mM NaCl, pH 8.0 (buffer B) and 10 mM imidazole. The lysate was loaded onto the Ni-NTA column equilibrated with buffer B containing 10 mM imidazole. The column was washed with 20 mM imidazole in buffer B and eluted with 250 mM imidazole in buffer B. The concentrated protein was further purified by gel filtration, concentrated again, dialyzed, lyophilized, and stored at -80 °C as described earlier. Equilibrium Binding Studies. The procedures for the equilibrium binding studies of DHNAs were essentially the same as previously described for the similar studies of 6- hydroxymethyl-7,8-dihydropterin pyrophosphokinase using a Spex F luoroMax-2 fluorometer (25, 26). Briefly, proteins and ligands were all dissolved in 100 mM Tris- HCl, pH 8.3, and the titration experiments were performed in a single cuvette at 24 °C. For the NP, MP, and HPO experiments, a DHNA solution was titrated with a stock solution of one of the ligands. Fluorescence intensities were measured at an emission wavelength of 446 nm with a slit of 5 nm. The excitation wavelength and slit were 400 nm and 1 nm, respectively. A set of control data was obtained in the absence of the protein. The data set obtained in the absence of the protein was then subtracted from the 40 corresponding data set obtained in the presence of the protein after correcting inner filter effects. The Kd value was obtained by nonlinear least square fitting of the titration data as previously described (25). To determine the Kd values for HP, a solution containing 1 uM HP was titrated with the proteins. The fluorescence of HP was measured at an emission wavelength of 430 nm and an excitation wavelength of 330 nm. The emission and excitation slits were both 5 nm. A control titration experiment was performed in the absence of HP. The control data set obtained in the absence of HP was subtracted from the corresponding data set obtained in the presence of HP. The [(4 values were obtained by nonlinear least square fitting of the titration data as previously described (25). Stopped-F low Analysis. Stopped-flow experiments were performed on an Applied Photophysics SX.18MV-R stopped-flow spectrofluorometer at 25 °C. One syringe contained the protein (SaDHNA or EcDHNA), and the other contained NP, MP, HP or HPO. The protein concentrations were 1 or 2 M, and the ligand concentrations ranged 5-60 uM. All concentrations were those after the mixing of the two syringe solutions. Fluorescence traces for NP, MP and HPO were obtained with an excitation wavelength of 360 nm and a filter with a cutoff of 395 nm for emission. Fluorescence traces for HP were obtained with an excitation wavelength of 330 nm and the same filter for emission. Apparent rate constants were obtained by nonlinear squares fitting of the data to a single exponential equation and were re-plotted against the ligand concentrations. The association and dissociation constants were obtained by linear regression of the apparent rate constants vs. ligand concentration data. 41 Quench-F low Analysis. Quench-flow experiments were carried out on a KinTek RQF-3 rapid quench-flow instrument. One syringe was loaded with a protein solution (SaDHNA or EcDHNA), and the other loaded with a substrate solution (DHNP or DHMP). All components were dissolved in a buffer containing 100 mM Tris-HCl, 1 mM EDTA, and 5 mM DTT, pH 8.3. For the forward reaction with DHNP or DHMP as the substrate, the enzyme concentrations were 15-20 M, and the substrate concentrations were 10, 20, and 30 uM, all referred to those immediately after the mixing of the two syringe solutions. For the reverse reaction, the enzyme (SaDHNA or EcDHNA) was 10 uM, HP was 100 uM, and GA ranged from 1 to 100 mM. All reactions were initiated by mixing of the two solutions, one containing the enzyme and the other the substrate(s), and quenched with 1 N HCl. The quenched reaction mixtures were processed as previously described (15). Briefly, the reaction mixtures (115 pl each) were mixed with 50 uL 1% 12 (w/v) and 2% (w/v) K1 in 1 N HCl for 5 min at room temperature to oxidize the pterin compounds. Excess iodine was reduced by mixing with 25 uL 2% ascorbic acid (w/v). The samples were then centrifuged at room temperature for 5 min using a microcentrifuge. The oxidized reactant and products in the supematants were separated by HPLC using a Vydac RP18 column. The column was equilibrated with 20 mM NaH2PO4 made with MilliQ water and eluted at the flow rate of 0.8 mL/min with the same solution. The oxidized reactant and products were quantified by online fluorometry with excitation and emission wavelengths of 365 and 446 nm, respectively. The quench- flow data were analyzed by global fitting using the program DYNAF IT (27) according to Scheme 1. 42 NMR Spectroscopy. NMR measurements were made at 25 °C with a Varian lnova 600 spectrometer. The initial NMR sample contained 2 mM DHNP and 1 mM tris(2- carboxyethyl) phosphine (TCEP) in 50 mM sodium phosphate buffer, pH 8.3 (pH meter reading without correction for deuterium isotope effects), made with D2O. The reaction was initiated with 3 uM SaDHNA. NMR spectra were recorded before and after the addition of the enzyme. A spectrum of DHMP was also acquired for comparison. The spectral width for the NMR data was 8000 Hz with the carrier frequency at the HDO resonance. The solvent resonance was suppressed by presaturation. Each FID was composed of 16 k data points with 16 transients. The delay between successive transients was 6 s. The time domain data were processed by zero-filling to 32 k points, multiplication with a 90°-shifted sine bell function, and Fourier transformation. Chemical shifts were referenced to the internal standard sodium 2-dimethyl-2- silapentane-S-sulfonate sodium salt (DSS). The relative proton populations were calculated based on the integrals of their NMR signals. RESULTS NMR Analysis. Although it is reasonable that the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction as described earlier (Figure 2.1), it is also possible that the epimerase reaction follows an alternative mechanism, i.e., the deprotonation and re-protonation of 2’-carbon. The alternative reaction can be initiated by deprotonation of 1’-carbon and protonation of 5—nitrogen to form an enol 43 intermediate, which can turn into a keto intermediate by tautomerization for the subsequent deprotonation and re-protonation of 2’-carbon. Whether the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction or the mechanism of deprotonation and re-protonation of 2’-carbon can be tested by NMR. The key difference between the two reaction mechanisms is that 2’-proton is always attached to 2’-carbon if the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction (Figure 2.1), while it has to be extracted by a base if the epimerase reaction follows the mechanism of deprotonation and re-protonation of 2’-carbon. Therefore, when the reaction is run in D2O, the 2’-proton occupancy will change if the epimerase reaction involves the deprotonation and re-protonation of 2’-carbon but will not change if it follows the same reaction intermediate as that of the aldolase reaction. The proton occupancy can be quantified by NMR. It was noticed in this context that the 6-hydroxymethyl group of the aldolase reaction product HP can be significantly deuterated (at least half of the —CH2- protons of the hydroxymethyl group) if the reaction occurs in D20 (24). The result of such an experiment is shown in Figure 2.2. The NMR signals were assigned based on their multiplicity patterns, decoupling experiments, and comparison with the NMR spectrum of authentic DHMP (the top spectrum in Figure 2.2). As shown in Figure 2.2, the NMR signals of all 2’- and 3’-protons of DHNP and DHMP are well separated, except those of the 3’Hb protons of the two compounds, which are overlapping. The proton occupancy at the 2’-position of the newly formed DHMP could be quantified by comparing the integrals of the 2’H and 3’Ha NMR signals of DHMP, because 3’-protons do not participate in the chemical reaction in either mechanism and cannot be replaced with deuterons. The result showed that the intensities of the 2’H and 44 Figure 2.2: The SaDHNA-catalyzed reactions in D20 monitored by N MR. The bottom spectrum was obtained before the addition of the enzyme and the middle three spectra were obtained 18, 35, and 70 min after the addition of the enzyme. The top spectrum is that of DHMP for comparison. Only the NMR signals of 2’ and 3’ protons of DHNP and DHMP are shown. The chemical structures of DHNP and DHMP are also shown at the top with atom numbering labeled for DHNP. For clarity, the NMR signals of the aldolase reaction products HP and GA are not shown. 45 3'b 3'b OH H OH N 2' N\ 5 2' HN3 I 5\ 1, OH H" l 1. 5 0H *1 8 OH H “Mk" N OH H ”2" N u 3'3 2 H 3Ia DHNP DHMP 31‘“ 3'Hb 2H DHMP 70mm 35 min 18mm DHNP 2H 3Ha 3Hb IlllllllllllllIIUIIIIIIlllllllllllll'lll 385 380 335 330 315 310 335 ”MI 46 3’Ha NMR signals were the same throughout the time course of the reaction (18, 35, and 70 min). The 1:1 intensities of the 2’H and 3’Ha NMR signals indicated a 100% proton occupancy at 2’-position, strongly suggested that there is no deprotonation and re- protonation at 2’-carbon and the epimerase reaction follows the aldol chemistry. Is the DHNA-catalyzed reaction reversible? Although aldolase-catalyzed reactions are generally reversible, the DHNA-catalyzed reaction was shown previously to be irreversible (12). However, it was noticed that the E. coli enzyme preparation used in the experiment had a low activity and furthermore, the GA concentration (150 uM) was rather low, especially considering that GA exists in various forms in solution and only a small fraction is in the correct form for the reaction (28, 29). To further investigate the issue of the reversibility of the DHNA-catalyzed reaction, we ran the reverse reaction with our recombinant enzymes and high concentrations of GA. One such result obtained with SaDHNA is shown in Figure 2.3. Clearly, the SaDHNA-catalyzed reaction was reversible. Furthermore, the reverse reaction was rather rapid in the presence of SaDHNA. The apparent Km for GA obtained by varying GA at a fixed HP concentration (100 uM) was ~10 mM. The EcDHNA-catalyzed reaction was also reversible, but the reverse reaction catalyzed EcDHNA was much slower than that catalyzed by SaDHNA (data not shown). Equilibrium Binding Studies. Since the epimerase reaction uses the same reaction intermediate as that of the aldolase reaction and the aldolase reaction is reversible, we can draw a unified kinetic scheme for the DHNA-catalyzed reactions as shown in Scheme 1, where A, B, I, P, and Q represent DHNP, DHMP, the reaction intermediate, HP, and glycolaldehyde, respectively. 47 Figure 2.3: Reverse reaction catalyzed by SaDHNA. The initial reaction mixture in 100 mM Tris-HCI, pH 8.3, contained 100 M HP and 20 mM GA. The reaction was initiated with 10 uM SaDHNA at 25 °C, quenched with 1 N HCl, and processed as described in the Experimental Procedures section. The HPLC chromatograms only show the oxidized pterin substrate (HPO) and products (NP and MP), because GA has no fluorescence. 48 Fluorescence 140 120 100 80 l l W3 N. I l MP\ _ Reaction time: 4.8 s 2 Al \_ Reaction time: 2. 4 s J \/\T____.L _ Reaction time: 0.96 s \A—r‘”[ Reaction time: 0.48J\~___ _[ Reaction time: 0 s J l _ | . ‘1 0 5 1 0 1 5 Retention time (s) 49 Scheme 1 E +A—E'1—‘EAJ—EHfi—EBéE + B K1 K2 K5 k-6 ..II. EPQ “ll“ E+P+Q The major goal of this work was to determine the rate constants of the individual steps of the reactions. Our strategy to achieve this goal was a comprehensive one, involving the measurements of both equilibrium and kinetic constants of the physical steps by equilibrium and stopped-flow fluorometric analysis and the determination of the rate constants of the chemical steps by quench-flow analysis of both forward and reverse reactions. We first measured the dissociation constants by fluorometry. A typical fluorometric titration curve is shown in Figure 2.4. The results are summarized in Table 2.1. To facilitate the purification of SaDHNA, we engineered a His-tag at the N-terrninal of the enzyme. The binding properties of the His-tagged and untagged enzymes were essentially the same (data not shown), and the binding data for SaDHNA in Table 2.1 are those of the His-tagged enzyme. NP, MP, and HPO are the oxidized forms of DHNP, DHMP, and HP, respectively. The only difference between the two sets of the pterin compounds is that between C7 and N8 is a single bond in the reduced pterins, but a double bond in the oxidized pterins. Consequently, there is a hydrogen atom attached to N8 in the reduced pterins and the NH group can serve as a hydrogen bond donor, while in the oxidized pterins, there is no hydrogen attached to N8 and it can only serve as a 50 Figure 2.4: Binding of NP to SaDHNA at equilibrium. A 2 mL solution containing 15 IIM SaDHNA in 100 mM Tris-HCl, pH 8.3 was titrated with NP by adding aliquots of a 1.94 mM NP stock solution at 24 °C. The final enzyme concentration was 14 IIM. The top axis indicates the NP concentrations during the titration. A set of control data was obtained in the absence of the enzyme and was subtracted from the corresponding data set obtained in the presence of the enzyme. The solid line was obtained by nonlinear least-squares regression as previously described (25 ). 51 0 191! 38.0 56.5 74.6 92.4 110 127 144 (IIM) . l . . . , . I .41 . 120000- 4 100000- 80000- J 60000- 4 40000- Fluorescence 20000 1 l o- 1 ' —l T I ' l -20000 fi -20 fi'fi'l 0 20 40 ...f.,.,.,... 60 80 100 120 140 160 180 NP(ul) 52 Table 2.1: Dissociation Constants of S. aureus and E. coli DHNAs Measured by Equilibrium Binding Experiments SaDHNA“ EcDHNA Kw, (0M) 18i2 0.772006 KM...) (0M) 13¢] 2.6:t0.06 K...) (uM) 2410.2 O.43i0.04 1rd,...» (0M) 24i0.2 O.lO:l:0.007 aThe chemical structures of the measured compounds are as follows. 0 0H 0 9H 0 N : N ’ N HN \ 0H HN \ ; OH HN \ OH )\\ I / OH )\\ I / 6H )\\ l / H2N N N H2N N N H2N N N NP MP HPO bSaDHNA has a His-tag at the N-terminus. 53 hydrogen bond acceptor. NP, MP, and HPO are all DHNA inhibitors. The results of the equilibrium binding studies showed that in comparison with EcDHNA, SaDHNA has significantly higher Kd values for the measured pterin compounds, particularly HPO, whose the Kd value for SaDHNA was 240 times that for EcDHNA. Furthermore, while the Kd values of SaDHNA for the reduced and oxidized pterin compounds (HP and HPO respectively) were the same, the Kd value of EcDHNA for the reduced pterin compound (the product HP) was higher than that for the oxidized pterin compound (the oxidized product HPO). Finally, the K2 value of SaDHNA for NP was slightly higher than that for MP, while the Kd value of EcDHNA for NP is lower than that for MP. Stopped-F low Analysis. We then measured the rate constants of the physical steps of the reaction by stopped-flow fluorometric analysis. Because glycolaldehyde (GA) has a very low affinity for the enzymes (data not shown) and exists in solution in multiple forms, of which the correct form for the reaction is a minor one (28, 29), we focused our analysis of product binding and dissociation on HP. Because DHNP and DHMP undergo chemical reactions in the presence DHNA, we measured the binding and dissociation of the structurally-related DHNA inhibitors NP and MP. To assess the differences in the rate constants of the reduced and oxidized pterins, we also measured the association and dissociation rate constants of HPO and compared them with those of HP. A typical result of the stopped-flow analysis is shown in Figure 2.5. The rate constants measured by the stopped-flow experiments are summarized in Table 2.2, where In and k.I are the association and dissociation rate constants, respectively. The Kd values calculated as k. I/kl were in excellent agreement with those measured by equilibrium binding studies 54 Figure 2.5: Stopped-flow analysis of the binding of HPO to EcDHNA. The concentration of EcDHNA was 0.2 IIM, and the concentrations of HPO were 1, 2, 4 and 8 ILM for traces 1, 2, 3, and 4, respectively. All concentrations were those immediately after the mixing of the two syringe solutions. Both EcDHNA and HPO were dissolved in 100 mM Tris-HCl, pH 8.3. The fluorescent signals were rescaled so that they could be fitted into the figure with clarity. The solid lines were obtained by nonlinear regression as described in the Experimental Procedures section. The inset is a replot of the apparent rate constants vs. the HPO concentrations. The solid line was obtained by linear regression. 55 Fluorescence 0.05 0.04- 0.03- 0.02- 0.01 - 0.001 HPO (uM) V‘w'rvv ..VV .w—rv- . ' , IA Iii-r [gumbo w .c Iva-gm w '0!" v7 ”33‘ ‘fl‘li'l'l" ' V I I I ' I I I o 5 10 15 20 Time (s) 56 Table 2.2: Association and Dissociation Rate Constants of S. aureus and E. coli DHNAs Measured by Stopped-flow Experiments SaDHNAa EcDHNA kl k-. de kl k.. [if (uM"S") 0“) (PM) (uM"s") 0“) (uM) NP 0.24d:0.01 4.5i0.1 19 0.32i0.02 0.29i0.03 0.88 MP 0.29:t0.02 4.2:t0.2 15 0.26i0.01 0.58i0.03 2.3 HP 0.47zt0.04 13:1:1 28 0.65:1:0.08 0.26d:0.02 0.4 HPO 0.45zt0.02 10i0.5 24 0.55:1:0.04 0.062i0.006 0.1 1 aSaDHNA has a His-tag at the N-terminus. bThe Kd values were calculated as k-l/k1. 57 (Table 2.1). The results showed that the association rate constants for NP and MP are very similar and slightly lower than those for HP and HPO, which are very similar. This phenomenon is presumably related to the sizes of the molecules. NP and MP are the same size and are slightly larger than HP and HPO. Furthermore, the results also showed that for SaDHNA, the association and dissociation rate constants of the reduced pterin HP are the same as those of the oxidized pterin (HPO), in accordance with the same Kd value for the two pterin compounds. 0n the other hand, for EcDHNA, the association rate constants for HP and HPO are essentially the same, but the dissociation constant of HP is larger than that of HPO, in agreement with a larger Kd value for HP. Finally, the higher Kd values are all mainly due to the higher dissociation rate constants. Quench-Flow Analysis. The rate constants of the chemical steps were measured by quench-flow experiments. We ran the forward reaction (the formation of HP) using both DHNP and DHMP as the substrates and the reverse reaction (the formation of DHNP and lDHMP) with HP and GA. For the forward reaction, three concentrations each for DHNP and DHMP were used. For the reverse reaction, the concentration of HP was fixed, and eight concentrations of GA were used for the SaDHNA-catalyzed reaction and six concentrations of GA for the EcDHNA-catalyzed reaction. Each reaction generated three curves, one each for DHNP, DHMP, and HP. The multitude of the quench-flow data was then fitted globally to Scheme 1 by nonlinear least squares regression using the program DYNAF IT (27). The initial values for the physical steps were from the stopped- flow analysis described in the previous section. Typical results of the forward reaction are shown in Figures 2.6 and 2.7 for the SaDHNA- and EcDHNA-catalyzed reactions, respectively, and for clarity, only the formation of HP was plotted. The results of the 58 Figure 2.6: Quench-flow analysis of the SaDHNA-catalyzed reaction. Data 1, 2, and 3 were obtained with DHNP as the substrate. Because the commercial DHNP contained a minute amount of DHMP, the reaction mixtures contained both DHNP and DHMP. The concentrations of DHNP and DHMP for data 1, 2, and 3 were 29.7 and 0.3, 19.8 and 0.2, 9.9 and 0.1 IIM, respectively. Data 4, 5, and 6 were obtained with DHMP as the substrate. The DHMP concentrations were 10, 20, and 30 IIM, respectively. The enzyme concentration was 20 IIM for all reactions. All concentrations were those immediately after the mixing of the two syringe solutions. The buffer contained 100 mM Tris-HCl, 5 mM DTT, pH 8.3. The solid lines were obtained by global nonlinear least squares regression using the program DYNAF IT (2 7). For clarity, the changes in the concentrations of DHNP and DHMP were not plotted. 59 a, . ..//o/ 5.0 4.0 3.0 Time (s) 10.0 ' 2.0 r 0 60 Figure 2.7: Quench-flow analysis of the EcDHNA-catalyzed reaction. The reaction conditions and substrate concentrations were the same as those described in the legend for Figure 2.6. The EcDHNA concentration was 15 IIM for all data sets. The solid lines were obtained by global nonlinear least squares regression using the program DYNAFIT. For clarity, the changes in the concentrations of DHNP and DHMP were not plotted. 61 .12 34 2.5 0.5 120 10.0 - 2.0 1.5 "I”Ime (s) 1.0 62 quench-flow analysis are summarized in Figure 2.8. For SaDHNA, the epimerase activity is insignificant in comparison with its aldolase activity, only a small fraction of the aldolase activity, the rate-limiting step in the formation of HP is the generation of the reaction intermediate, and the reverse reaction is faster than the forward reaction. For EcDHNA, in contrast, the epimerase activity is highly significant, comparable to the aldolase activity, the rate-limiting step in the formation of HP is the product release, and the reverse reaction is much slower than the forward reaction. DISCUSSION Despite its fundamental significance as a unique aldolase and its biological significance as an attractive target for developing antimicrobial agents, how the enzyme catalyzes both the aldolase and epimerase reactions are largely unknown beyond the crystal structures (11, I6, 30, 31). While it is reasonable to assume that the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction, one cannot exclude a priori the alternative mechanism of deprotonation and re-protonation of 2-carbon. Our NMR analysis of DHMP generated in the reaction in D20 clearly indicated that there is no deuteration of 2-carbon of the epimerase product. In contrast, the 6- hydroxymethyl group of the aldolase product HP is deuterated to the extent of having at least one equivalent of deuterons added to the 6-hydroxymethyl carbon (24). Obviously, the lack of deuteration of the 2’-carbon of DHMP is not due to the lack of deuterons, rather it suggests that the epimerase reaction follows the aldol chemistry and the 2’- proton is not extracted during the course of the reaction. The NMR data strongly supports 63 Figure 2.8: Summary of the kinetic constants for the SaDHNA-catalyzed (top panel) and EcDHNA-catalyzed (lower panel) reactions. The chemical steps are highlighted with blue ovals, and the aldolase and epimerase activities with blue arrows. 64 0.24 ”wet 033 s" 020 a" 42::1 Sa + DHNP-—-Sa. Dl-ND—I-Sa. l-—-=Sa.DlW-—=Sa + Dl-M’ 4.5 a 1 1.6 a“ 0.076 6" 029105134 130 a“ 1003-1 Sal-PEA 0.54 14 a" IIM’18'1 Sa+l-IP+GA Aldolase 0.16 a“ 6.0 a" Ec.l-P.GA 0.65 0.28 s" “"134 EC + 11’ + GA Aldolase 65 the hypothesis that the aldolase and epimerase activities follows the same reaction intermediate as depicted in Figure 2.1. Because DHNA catalyzes both aldol and epimerization reactions and the epimerization product DHMP can also be converted to the aldol reaction product HP, it is particularly important to determine the rate constants for elementary steps if one intends to determine how the enzyme catalyzes the two reactions. Furthermore, steady-state kinetic analysis is insufficient for DHNA, because the steady-state kinetic parameters cannot adequately describe the two reactions catalyzed by the enzyme and the formation of DHMP will be underestimated because of its conversion to HP. HauBmann and coworkers previously determined the steady-state kinetic constants for EcDHNA (15). According to the steady-state kinetic data, the epimerase activity is 1/6 of the aldolase activity, which significantly underestimates the epimerase activity of EcDHNA (see Figure 2.8, lower panel). Furthermore, the kc... values for the aldolase and epimerase activities are significantly lower than the rate constants of the chemical steps. A critical issue in the kinetic analysis is whether the reaction is reversible or not. Although in general aldolase-catalyzed reactions are readily reversible, it was shown earlier that DHNA was an exception and the DHNA-catalyzed reaction is apparently irreversible. The apparent irreversibility is probably due to the low activity of the enzyme preparation used in the experiment, the low concentration of GA, and the low reaction rate of the EcDHNA-catalyzed reverse reaction. With pure recombinant enzymes and high concentrations of GA, it is clear that DHNA-catalyzed reaction is reversible. In fact for SaDHNA, the reverse reaction is much faster than the forward reaction. Our strategy for determining the rate constants of individual steps is a comprehensive one. The 66 philosophy behind the strategy is to isolate the different steps of the reaction whenever possible and design experiments to determine rate constants for the specific steps. Stopped-flow fluorometry is rapid and suitable for the physical steps of the enzymatic reaction, but is not suitable for the chemical steps because of the lack of significant optical changes in the chemical steps. 0n the other hand, quench-flow analysis is laborious but provides an accurate measurement of the rates of the chemical steps. Because the reaction is reversible, it can be run in three directions with DHNP, DHMP, or HP and GA as the substrates. One can then derive a complete set of rate constants for the enzymatic reaction by the global analysis of the multitude of data. The determination of a complete set of kinetic constants for both SaDHNA and EcDHNA provides a firm basis for dissecting their catalytic mechanisms. Our equilibrium and kinetic data show that SaDHNA and EcDHNA have significantly different binding and catalytic properties, in accordance with the significant sequence differences between the two enzymes. EcDHNA is different from SaDHNA biochemically in several aspects. (1) EcDHNA has much higher affinities for the substrate, products, and inhibitors measured in this work, particularly for HPO. (2) EcDHNA has a much higher epimerase activity than SaDHNA. (3) The rate-limiting step in the forward reaction (the formation of HP) is the product release for EcDHNA but is the formation of the reaction intermediate for SaDHNA. (4) The reverse reaction is very slow with EcDHNA but very fast with SaDHNA. The marked differences in the ligand- binding properties of SaDHNA and EcDHNA, which must stem from the significant differences in the structures of their active sites, suggest that it may be possible to develop antimicrobial agents specifically against DHNA from S. aureus or E. coli. 67 Indeed, we have developed potent inhibitors for SaDHNA or EcDHNA (F elczak et al., unpublished). Because many DHNAs from Gram-positive and Gram-negative bacteria are highly homologous within their own groups but significantly different between the two groups, it may be possible to develop antimicrobial agents specifically against Gram- positive or Gram-negative bacteria by targeting respective DHNAs. 68 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) REFERENCES Yao, L. 8., Yan, H. G., and Cukier, R. I. (2006) Mechanism of dihydroneopterin aldolase: A molecular dynamics study of the apo enzyme and its product complex. J. Phys. Chem. B 110, 1443-1456. World Health Organization (1999) World Health Organization Report on Infectious Diseases: Removing Obstacles to Healthy Development. World Health Organization, Geneva. Walsh, C. (2003) Where will new antibiotics come from? Nature Rev. Microbiol. 1, 65-70. Cohen, M. L. (2000) Changing patterns of infectious disease. Nature 406, 762- 767. Murray, B. E. (1997) Antibiotic resistance. Ad. Intern. Med. 42, 339-367. Projan, S. J. (2003) Why is big pharma getting out of antibacterial drug discovery? Curr. Opin. Microbiol. 6, 427-430. ' Spellberg, 3., Powers, J. H., Brass, E. P., Miller, L. G., and Edwards, J. E. (2004) Trends in antimicrobial drug development: Implications for the future. Clin. Infect. Dis. 38, 1279-1286. Wenzel, R. P. (2004) Business and medicine - the antibiotic pipeline - challenges, costs, and values. N. Engl. J. Med. 351, 523-526. Barrett, C. T., and Barrett, J. F. (2003) Antibacterials: Are the new entries enough to deal with the emerging resistance problems? Curr. Opin. Biotechnol. 14, 621- 626. Blakley, R. L., and Benkovic, S. J. (1984) in Folates and pterins, John Wiley & Sons, New York. Sanders, W. J., Nienaber, V. L., Lerner, C. G., McCall, J. 0., Merrick, S. M., Swanson, S. J., Harlan, J. E., Stoll, V. S., Stamper, G. F., Betz, S. F ., Condroski, K. R., Meadows, R. P., Severin, J. M., Walter, K. A., Magdalinos, P., Jakob, C. G., Wagner, R., and Beutel, B. A. (2004) Discovery of potent inhibitors of dihydroneopterin aldolase using crystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization. J. Med. Chem. 47, 1709-1718. 69 (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) Mathis, J. B., and Brown, G. M. (1970) The biosynthesis of folic acid XI. Purification and properties of dihydroneopterin aldolase. J. Biol. Chem. 245, 3015-3025. Horecker, B. L., Tsolas, 0., and Lai, C.-Y. (1975) Aldolases, in The enzymes (Boyer, P. D., Ed.) pp 213-258, Academic Press, San Diego. Allen, K. N. (1998) Reactions of enzyme-derived enamines, in Comprehensive biological catalysis (Sinnott, M., Ed.) pp 135-172, Academic Press, San Diego. HauBmann, C., Rohdich, F ., Schmidt, E., Bacher, A., and Richter, F. (1998) Biosynthesis of pteridines in Escherichia coli - structural and mechanistic similarity of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase. J. Biol. Chem. 273, 17418-17424. Hennig, M., D'Arcy, A., Hampele, I. C., Page, M. G. P., Oefner, C., and Dale, G. E. (1998) Crystal structure and reaction mechanism of 7,8- dihydroneopterin aldolase from Staphylococcus aureus. Nature Struct. Biol. 5, 357-362. Luo, Y., Samuel, J ., Mosimann, S. C., Lee, J. E., Tanner, M. E., and Strynadka, N. C. J. (2001) The structure of l-ribulose-S-phosphate 4-epimerase: An aldolase- like platform for epimerization. Biochemistry 40, 14763-14771. Samuel, J ., Luo, Y., Morgan, P. M., Strynadka, N. C. J., and Tanner, M. E. (2001) Catalysis and binding in l-iibulose-S-phosphate 4-epimerase: A comparison with l-fuculose—l-phosphate aldolase. Biochemistry 40, 14772-14780. Salzmann, M., Pervushin, K., Wider, G., Senn, H., and Wiithrich, K. (2000) NMR assignment and secondary structure determination of an octameric 110 kDa protein using TROSY in triple resonance experiments. J. Am. Chem. Soc. 122, 7543-7548. Wider, G., and Wiithrich, K. (1999) NMR spectroscopy of large molecules and multimolecular assemblies in solution. Curr. Opin. Struct. Biol. 9, 594-601. Riek, R., Pervushin, K., and Wilthrich, K. (2000) TROSY and CRIN EPT: NMR with large molecular and supramolecular structures in solution. Trends Biochem. Sci. 25, 462-468. Pervushin, K. (2000) Impact of transverse relaxation optimized spectroscopy (TROSY) on NMR as a technique in structural biology. Q. Rev. Biophys. 33, 161- 197. Deng, H., Callender, R., and Dale, G. E. (2000) A vibrational structure of 7,8- dihydrobiopterin bound to dihydroneopterin aldolase. J. Biol. Chem. 275 , 30139- 30143. 70 (24) (25) (26) (27) (28) (29) (30) (31) Illarionova, V., Eisenreich, W., Fischer, M., Haussmann, C., Romisch, W., Richter, G., and Bacher, A. (2002) Biosynthesis of tetrahydrofolate - stereochemistry of dihydroneopterin aldolase. J. Biol. Chem. 27 7, 28841-28847. Li, Y., Gong, Y., Shi, G., Blaszczyk, J ., Ji, X., and Yan, H. (2002) Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6- hydroxymethyl-7,8-dihydropterin pyrophosphokinase. Biochemistry 41, 8777- 8783. Shi, G., Gong, Y., Savchenko, A., Zeikus, J. G., Xiao, B., Ji, X., and Yan, H. (2000) Dissecting the nucleotide binding properties of Escherichia coli 6- hydroxymethyl-7,8-dihydropterin pyrophosphokinase with fluorescent 3'(2)'-o- anthraniloyladenosine 5'-triphosphate. Biochim. Biophys. Acta 1478, 289-299. Kuzmic, P. (1996) Program DYNAF IT for the analysis of enzyme kinetic data: Application to HIV proteinase. Anal. Biochem. 23 7, 260-273. Yaylayan, V. A., Harty-Majors, S., and Ismail, A. A. (1998) Investigation of the mechanism of dissociation of glycolaldehyde dimer (2,5-dihydroxy-1,4-dioxane) by ftir spectroscopy. Carbohydr. Res. 309, 31-38. ' Collins, G. C. S., and George, W. O. (1971) Nuclear magnetic resonance spectra of glycolaldehyde. J. Chem. Soc. B, 1352-1355. Bauer, 8., Schott, A. K., Illarionova, V., Bacher, A., Huber, R., and Fischer, M. (2004) Biosynthesis of tetrahydrofolate in plants: Crystal structure of 7,8- dihydroneopterin aldolase from arabidopsis thaliana reveals a novel adolase class. J. Mol. Biol. 339, 967-979. Goulding, C. W., Apostol, M. 1., Sawaya, M. R., Phillips, M., Parseghian, A., and Eisenberg, D. (2005) Regulation by oligomerization in a mycobacterial folate biosynthetic enzyme. J. Mol. Biol. 349, 61-72. 71 CHAPTER 3: MECHANISM OF DIHYDRONEOPTERIN ALDOLASE: A POINT MUTATION CONVERTS THE ENZYME TO A COFACTOR—INDEPENDENT OXYGENASE ABSTRACT Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8- dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. Substitution of a conserved tyrosine residue at the active site of DHNA by phenylalanine converts the enzyme to a cofactor-independent oxygenase, which generates mainly dihydroxanthopterin (DHXP) rather than HP. DHXP is generated via the same enol intermediate as in the wild-type enzyme-catalyzed reaction, but this species undergoes an oxygenation reaction to form DHXP. The conserved tyrosine residue plays only a minor role in the formation of the enol reaction intermediate, but a critical role in the protonation of the enol intermediate to form HP. 72 INTRODUCTION Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8- dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway, one of principal targets for developing antimicrobial agents (1). Like other enzymes such as dihydropteroate synthase and dihydrofolate reductase in the folate pathway (2, 3), DHNA is an attractive target for developing antimicrobial agents (4). DHNA is also a unique aldolase in that it requires neither the formation of a Schiff base between the substrate and the enzyme nor metal ions for catalysis (5) and in addition to the aldolase reaction, DHNA also catalyzes the epimerization at the 2’-carbon of DHNP to generate 7,8-dihydromonapterin (DHMP, Figure ' 1.3) (6). Interestingly, DHNAs from Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA), representatives of DHNAs from Gram-positive and Gram-negative bacteria respectively, have significant differences in binding and catalytic properties. One of the conserved residues at the active site of DHNA is a tyrosine residue, Y54 in SaDHNA and Y53 in EcDHNA. According to the crystal structure of SaDHNA in complex with HP (7), the pterin ring is stacked with the phenol ring of Y54 (Figure 1.7). The hydroxyl group of the phenol ring of Y54 is hydrogen bonded to the amino group of the putative general base of K100 and the hydroxyl of the 6-hydroxymethyl group of the bound HP. To investigate the role of the conserved tyrosine residue in catalysis, we replaced Y54 of SaDHNA and Y53 of EcDHNA with a phenylalanine residue by site- directed mutagenesis. The single point mutations convert the aldolases to oxygenases, which generate mainly dihydroxanthopterin (DHXP) rather than HP. DHXP is generated 73 via the same enol intermediate as in the wild-type enzyme-catalyzed reaction, but this species undergoes an oxygenation reaction to form DHXP. The conserved tyrosine residue plays only a minor role in the formation of the enol reaction intermediate, but a critical role in the protonation of the enol intermediate to form HP. EXPERIMENTAL PROCEDURES Materials. 6-Hydroxymethylpterin (HPO), 6-Hydroxymethyl-7,8-dihydropterin (HP), 7,8-dihydro-D-neopterin (DHNP), 7,8-dihydro-L-monapterin (DHMP), D- neopterin (NP), L-monapterin (MP), 7,8-dihydroxanthopterin (DHXP), and 6-formyl-7,8- dihydropterin (F DHP) were purchased from Schircks Laboratories. Pfu DNA polymerase was purchased fi'om Stratagene. Other chemicals were from Sigma or Aldrich. Site-Directed Mutagenesis and Protein Purification. The mutants SaY54F and EcY53F, where Y54 of SaDHNA and Y53 of EcDHNA were replaced by Phe, respectively, were made by a PCR-based method using high-fidelity pfu DNA polymerase according to a protocol developed by Stratagene. The forward and reverse primers for making the SaY54F mutant were 5’-GTT ATTGATACAGTTCATTITGGTGAAGTGTTCGAAGAG G-3 ’ and 5 ’- CCTCTTCGAACA CTTCACCAAAATGAACTGTATCAATAAC-3, respectively. The forward and reverse primers for making the EcY53F mutant 5’- CGGATTGCCTCAGTTICGCTGACATTGCAGAAAC-3 ’ and 5 ’ - GTTTCTGCAATGTCAGCGAAACTGAGGCAATCCG-3’, respectively. The mutants were selected by DNA sequencing. In order to ensure that there were no unintended 74 mutations in the mutants, the entire coding sequences of the mutated genes were determined. The mutant proteins were purified as previously described (Wang et al., unpublished). Briefly, SaYSF was purified to homogeneity by a Ni-NTA column followed by a Bio-Gel A-0.5m gel column. EcY53F was purified by a DEAE-cellulose column followed by a Bio-Gel A-0.5m gel column. The purities of the protein preparations were checked by SDS-PAGE. The amino acid sequence of the purified proteins were confirmed by ‘top-down’ tandem mass spectrometry (8). The purified proteins were concentrated, dialyzed, lyophilized, and stored at -80 °C. Equilibrium Binding Studies. The procedures for the equilibrium binding studies of the DHNA mutants were essentially the same as described previously for the wild-type enzymes (Wang et al., unpublished). Briefly, proteins and ligands were all dissolved in 100 mM Tris-HCl, pH 8.3, and the titration experiments were performed in a single cuvette with a Spex FluoroMax-Z fluorometer at 24 °C. To determine the Kd values of the binding of NP and MP to SaY54F, a 2 mL solution containing 10 IIM SaY54F was titrated with a NP or MP stock solution of one of the ligands (NP, MP, or HPO). To determine the Kd values of the binding of HPO to SaY54F and the binding of NP, MP, and HP to EcY53F, a 2 mL solution containing 1 uM of one of the ligands (NP, MP, or HPO) was titrated with a stock solution of SaY54F or EcY53F. The K., values were obtained by nonlinear least square fitting of the titration data as previously described (9). Stopped-Flow Analysis. Stopped-flow experiments were performed on an Applied Photophysics SX.18MV-R stopped-flow spectrofluorometer at 25 °C as described previously for the wild-type enzymes (Wang et al., unpublished). Apparent rate constants 75 were obtained by nonlinear least squares fitting of the data to a single exponential equation and were re-plotted against the ligand concentrations. The association and dissociation constants were obtained by linear regression of the apparent rate constants vs. ligand concentration data. Kinetic assay. The kinetic experiments were performed manually. Both DHNP and DHNA were dissolved in a buffer containing 100 mM Tris-HCl, 1 mM EDTA, and 5 mM DTT, pH 8.3. The reactions were initiated by the addition of DHNA and stopped with 1 N HCl. The stopped reaction mixtures were processed and separated by HPLC as described (6). NMR Spectroscopy. NMR measurements were made at 25 °C with a Varian lnova 600 spectrometer. The initial NMR sample contained 1 mM DHNP and 1 mM tris(2- carboxyethyl) phosphine (TCEP) in 100 mM sodium phosphate buffer, pH 8.3 (pH meter reading without correction for deuterium isotope effects), made with 90% H20 and 10% D20. The reaction was initiated with 1 1.1M SaDHNA or 3 uM SaY54F. NMR spectra were recorded before and after the addition of the enzyme. The spectral width for the NMR data was 12000 Hz with the carrier frequency at the HDO resonance. The solvent resonance was suppressed by presaturation. Each F ID was composed of 16 k data points with 64 transients. The delay between successive transients was 1.7 s. The time domain data were processed by zero-filling to 32 k points, exponential multiplication (1 Hz), and Fourier transformation. Chemical shifts were referenced to the internal standard sodium 2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS). Mass Spectrometry (MS). All MS experiments were performed using a Thermo model LTQ linear ion trap mass spectrometer. The initial 300 pl samples contained 200 76 IIM DHNP and 4 uM SaDHNA or 10 IIM SaY54F in 5 mM ammonium carbonate, pH 8. Twenty ILL aliquots of the reaction mixtures were taken out at 1-min intervals and mixed with 80 uL of a solution containing 50% acetic acid and 1% methanol. The samples were further diluted with the same solution and then introduced to the mass spectrometer at a flow rate of 0.5 ILL/min by nanoelectrospray ionization (nESl). The nESI conditions used were: spray voltage 1.8 kV, heated capillary temperature 200 °C, capillary voltage -10 V, and tube lens voltage -50 V. Collision induced dissociation (CID) tandem mass spectrometry (MS/MS and M83) spectra were acquired at an activation q value of 0.25 using an isolation width of 1 or 2 Da (to monoisotopically isolate the precursor ion), a normalized collision energy of 30% to 40%, and an activation time of 30 ms or 300 ms. The values were chosen such that the gentlest conditions were used in order to completely dissociate the selected precursor ion (i.e., some precursor ions required a larger normalized collision energy and/or longer activation time). The MS, MS/MS and MS3 product ion spectra shown are the average of 60 individual mass analysis scans. Gas Chromatography/Mass Spectrometry (GC/MS) of GA. The reaction contained 200 IIM DHNP and 4 uM SaDHNA or 6.3 IIM SaY54F in 5 mM ammonium carbonate, pH 8.3. The reaction was stopped after 20 min by adding 1 N HCl and centrifuged for 5 min with a microcentrifuge. The supernatant collected was then 1yophilized overnight to dryness. The dried residue was incubated for l h with 10 ILL methoxyamine hydrochloride in pyridine (10 mg/mL) and N-(t-butyldimethylsilyl)-N— methyltrifluoroacetamide (MTBSTFA). GC/MS analyses were performed on an Agilent 5973 mass spectrometer coupled to a model 6890 gas chromatograph. All analyses were performed using 70 eV electron ionization. Separations were performed using a 30 meter 77 HP-SMS column and helium (36 cm/s) as carrier gas. The column temperature was programmed from 50 °C (4 min hold) to 285 °C at 10 degrees/min. HPLC Identification of Formic Acid. The reaction mixtures in 1 mL contained 200 IIM DHNP and 10 IIM SaDHNA or SaY54F in 100 mM HEPES-KOH, pH 8.3, and were mixed with 100 Ill 3.2 M H2804 after 20 min incubation at 24 °C. The solutions were centrifuged, filtered through a 0.45 micron syringe filter, and injected into a 7.8X300 mm Aminex HPX-87H column. Standard compounds, including formic acid, acetic acid, glycolic acid, glyceric acid, succinic acid, formaldehyde, glycolaldehyde, and glyceraldehyde, were treated the same way as the enzymatic reaction mixture. The column was eluted with 4 mM H2SO4 at a flow rate of 0.6 mL/min, and the elution was monitored by a Waters 2487 UV detector at 210 nm and a Waters 410 differential refractometer. Oxygen Consumption Assay. Oxygen consumption was measured using a Gilson 5/6H oxygraph with a Clark oxygen electrode at 25 °C. The initial samples in 1.8 mL contained 200 uM DHNP in 100 mM HEPES-KOH, pH 8.2. The reactions were initiated by 8 IIM SaDHNA or SaY54F at 25 °C. The turnover number for SaY54F was calculated using the slope of the initial linear part of the data obtained by linear regression. RESULTS Biochemical Analysis. Previously we established the thermodynamic and kinetic framework for the structure-function studies of SaDHNA and EcDHNA (Wang et al., unpublished). We performed the biochemical characterizations of the mutants SaY54F 78 and EcY53F using the same strategy. The binding steps were mimicked with the substrate and product analogues NP, MP, and HPO. The only difference between the substrate and product analogues and the corresponding substrate and products is that between C7 and N8 is a double bond in the analogues and a single bond in the substrate and products. NP, MP, and HPO are excellent substrate and product analogues (Wang et al., unpublished). The K: values were measured by equilibrium binding experiments using fluorometry. The association and dissociation rate constants were determined by kinetic binding experiments using stopped-flow fluorometry. Representative results are shown in Figures 3.1 and 3.2 for the equilibrium and kinetic binding experiments, respectively. The complete results are summarized in Table 3.1. For SaY54F, the mutation increased the affinities of the ligands for the enzyme by a factor of ~4-6. FOr EcY53F, the mutation increased the affinities of NP and MP by a factor of 4-6, but decreased the affinity of HPO by a factor of 4. The changes were mainly caused by the changes in the dissociation rate constants. The results suggested that neither Y54 of SaDHNA nor Y53 of EcDHNA is critically important for the binding of substrate or products. To our surprise, there was a dramatic drop in the total fluorescence intensity in the HPLC analysis of the reaction mixtures generated by either SaY54F or EcY53F as the reaction progressed, suggesting that the substrate was converted to a non-fluorescent compound at the chosen excitation and emission wavelengths and the mutant enzyme-catalyzed reactions generated different products. NMR Analysis. In order to identify the products of the mutant-catalyzed reactions, we analyzed the reaction mixtures by NMR. Representative spectra are shown in Figure 3.3, and the chemical shifts of selected protons of related compounds are summarized in 79 Figure 3.1: Binding of HPO to EcY53F at equilibrium. A 2 mL solution containing 1 uM HPO in 100 mM Tris-HCl, pH 8.3 was titrated with EcY53F by adding aliquots of a 60 1.1M EcY53F stock solution at 24 °C. The final HPO concentration was 0.93 M. The top axis indicates the EcY53F concentrations during the titration. A set of control data was obtained in the absence of HPO and was subtracted from the corresponding data set obtained in the presence of HPO. The solid line was obtained by nonlinear least-squares regression as previously described (9). 80 Fluorescence 220000 -I 200000 1 .1 180000 - ‘ 160000 - q 140000 - 1 120000 - d 100000 '1 d 80000 - 60000 1 4 40000 -20 ,. 60 EcY53F (0L) ,. 80 81 I I ' I l ' l ' 100 120 140 160 180 Figure 3.2: Stopped-flow analysis of the binding of MP to EcY53F. The concentration of EcY53F was 0.2 IIM, and the concentrations of MP were 1, 2, 4 and 8 IIM for traces 1, 2, 3, and 4, respectively. All concentrations were those immediately after the mixing of the two syringe solutions. Both EcY53F and MP were dissolved in 100 mM 100 mM Tris-HCl, pH 8.3. The fluorescent signals were rescaled so that they could be fitted into the figure with clarity. The solid lines were obtained by nonlinear regression as described in the Experimental Procedures section. The inset is a replot of the apparent rate constants vs. the MP concentrations. The solid line was obtained by linear regression. 82 0.240 0.235 - 0.230 3 0.225 1 0.220 5 0.215 5 0.210 -‘ 0.205 1 0.200 J Fluorescence 0.195. 2.5 2.0 - 1.51 kapp (5.1) 0.5 l 0.0 83 q — Table 3.1: Binding Constants of S. aureus and E. coli DHNAs and Y—+F Mutants SaDHNA SaY54F EcDHNA EcY53F NP Kd (0M) 1822 4.5209 0.772006 0.220.009 k1 (0M“s“) 0.242001 0.09920005 03220.02 0.2620003 k. (s!) 4.5201 0.442005 0.292003 006020.006 MP K. (M) 1321 3.7205 2.62006 0.402001 k1 (0M“s") 02920.02 0.1320003 0.262001 0.2820003 k.. (8") 4.2202 0.802009 0.582003 0.1120006 HPO K. (M) 2420.2 38209 01020007 0402002 k1 (0M"s") 04520.02 0.6820003 0.552004 07920.005 k.1 (5") 1020.5 2.92006 0.06220006 026200001 84 Figure 3.3: NMR analysis of the reactions catalyzed by SaDHNA (A) and SaY54F (B). The initial NMR sample contained 1 mM DHN P and 1 mM tris(2-carboxyethyl) phosphine (TCEP) in 100 mM sodium phosphate buffer, pH 8.3, made with 90% H20 and 10% D20. The reaction was initiated with 1 IIM SaDHNA (A) or 3 IIM SaY54F (B). 85 GA+ WAR-.. TCEP 2 HN 3 HaN 3 HbN+M __, GA 9.311110 M\* y, _,.,-.\__ MA __ M_ ___ 7H+1 11% 2 HM 3 HaM _2&,_k -..A- ”HA-m _2_____wam._.__2r\f\ “gnaw/"ANA. ._....__-.___.M... Emir) _.. “__A [\\_- 1x2 --. _- AW, .2222 m ._ M... .._.M_ FAME“- , k§7_min 2m-_flj\xv A ..... ,/\J\.2 22224-22” ,-,-_M--__-_.-JW\A “___“.nggflifl [\2 .--2. -22 W.,..A. _, l/‘x- , AIL,“~JV\NL,___M_21min x A- _. _ _,__, _,,__2___,,_ /‘\. _ __J‘K2_ ___JAM_ ___ ____2-___,_ $211119 -- .-- ____ .. __,_ _,._.._2_ “ ___ , _A“ _ _- f'1/‘\_ _MAL ....-.__..__._._ 0 min r, 7: vv ww—rfi‘ 3.9 3.8 3.7 3. 6 3. 5 ppm MR 1111. _, 11141- ._ 9’3 2582116 71/1 __ __llIAI _.__,M__§8mi_n .40 LA NM. -J.4:m0 .m2_mhzm-hm2_fl2.2@mm 22/502thth _ M 2.5 min -A-.-_-t111- _ -M_-__225...._12_n01 M M_.__:. 201.0 JhWLMMM _900 .12, v lvvvvr 3.9 3.8 3.7' 3.6 3.5 ppm 86 5.!" . l"’ math-ha Table 3.2. As shown in Figure 3.3, GA was generated by both the wild-type enzyme- and the mutant-catalyzed reactions. While the wild-type enzyme catalyzed reaction generated HP along with GA (Figure 3.3A), the mutant-catalyzed reaction generated little HP (Figure 3.38). The major product of the mutant-catalyzed reaction was a peak at 4.12 ppm. The NMR spectra of the reaction mixtures generated by the E. coli enzymes (data not shown) were very similar to those of the reaction mixtures generated by the S. aureus enzymes. Because both SaDHNA and SaY54F were saturated with the substrate DHNP under the experimental conditions, their turnover numbers (kw) could be calculated on the basis of the enzyme concentration, the initial substrate concentration, and the NMR peak intensities of the substrate and products. The calculated kw values for the formation of HP were 0.2 s'1 for SaDHNA and 3.4><104 s'1 for SaY54F. MS Analysis. The identity of the major product of the SaY54F-catalyzed reaction that gave rise to the 4.12 ppm peak in the NMR spectrum could not be determined by 1D proton NMR. In order to identify the reaction product, we analyzed the reaction mixtures generated by both the wild-type and the mutant enzymes by MS. As expected, the primary product generated by SaDHNA was different from that generated by SaY54F (Figures 3.4A and B). The mass spectrum of the reaction involving SaDHNA showed the formation of a product ion at m/z 196, while the spectrum of the reaction involving SaY54F showed the formation of a product ion at m/z I82. In order to determine the identity of the m/z 196 ion in Figure 3.4A, MS/MS and MS3 spectra were obtained and compared to those of the expected reaction product HP. As shown in Figure 3.5, the major ions observed by CID MS/MS of the ion at m/z 196 obtained from the SaDHNA-catalyzed reaction were identical to the major product ions 87 Table 3.2: Chemical Shifts of Selected Protons of Compounds Related to the Reactions Catalyzed by the Wild-Type and Mutant DHNAsa Compounds 7H 1 ’ H 2 ’H 3 ’Ha 3 ’Hb Others DHNP 4.22 (d) 4.22 (d) 3.90 (m) 3.80 (m) 3.66 (m) DHMP 4.22 (d) 4.27 (d) 3.94 (m) 3.72 (m) 3.66 (m) HP 4.18 (s) 4.18 (s) DHXP 4.12 (5) GA 3.50 (d) GA+TCEP 4.02 (m) alThe chemical shifts were from those of the commercial compounds in 50 mM sodium phosphate buffer, pH 8.3 (pH meter reading without correction for deuterium isotope effects), made with 90% H20 and 10% 020. The peak at 4.02 ppm was present only in the presence of both GA and TCEP. The 4.18 ppm peak of HP also belongs to the 6- hydroxymethyl group. 88 Figure 3.4: ESIl MS analysis of the mixtures generated by a 10 min reaction of SaDHNA (A) and SaY54F (B). The substrate, DHNP, is observed at m/z 256 in both spectra. The region from m/z 180-300 has been magnified (x5) for clarity. 89 % Relative Abundance % Relative Abundance x5 100‘ 177 l l 196 4 1 187 1 165 T 152 I66 “I 218 2 . 157 234 269 ' I71 182 239 256 j i 217 225 247 273 291 I 1 ‘94 y ‘ 1 1 279 1 1 1 1 263 ‘ I: l .L. l. H.811-.. .. ...L .... ... .AAJ‘...J1 .. “M. All.-.LL.Ll2 . l‘.. ...Lu.- ._.. 1.. ...A. 160 180 200 220 240 260 280 300 x5 1001 B 177 l87 4 165 152 166 I 1 182 . '57 j/ 1 234 256 269 l 171 1 247 - é 2‘7 225 ‘239 263 i . 194 9‘ 1 279 29‘ i. 1 J1. l. \111'. .1 .. u.L.. . .. ., ..I .1...J.. .14 ..“J... llll.1J..Ai...J. L -1.. . ., 1.. H... 160 180 200 220 240 260 280 300 Iniflz 9() Figure 3.5: Multistage tandem mass spectrometry identification of the m/z 196 product from the reaction of DHNP with SaDHNA. (A) CID MS/MS product ion spectrum of the ion at m/z 196 obtained from Figure 3.4A. (B) CID MS/MS product ion spectrum of the m/z 196 precursor ion obtained from a solution of commercially available HP. (C) CID MS3 product ion spectrum of the ion at m/z 178 in panel A. (D) CID MS3 product ion spectrum of the ion at m/z 178 in panel B. 91 NE CNN CCN CM: C3 C3 CN~ CC~ CC CC F k p p _ L [T1 _ r L p _ r _ _ _ a _. - _1 an. C__ mm— Vm_ on— Ch. ._o~:-:+§ - on. - _C_ nfiY n:2. Q rCC_ CNN CCN of C2 C3 CN_ CC# CC CC _ _ L p «_i _ . _ F L p _ _ _ . _ CC. .59: 62 - mo— mm. of- m -CE as CNN CCN of C3 C3 CN— CC_ CC CC — r w _ _ . I— . p 1_ m _ ~ _ r _ C2.— fi C: cm. mm— om~ 1 mn— ..of-:+§ _2 92 £2- o? D -2: CNN CCN C2 C2 C3 CN_ CC_ Cw CC _ _ _ _ _ f _ e r. _ h _ _ _ _ _ CC. .Eié 8. T mC_ fi mm— of- < .2: aouepunqv GADBlQH % aouepunqv aAuepH % 92 seen in the MS/MS product ion spectrum obtained from the standard (compare Figures 3.5A and B). The most abundant product ion in Figures 3.5A and B at m/z 178 is most likely due to the loss of water from the precursor ion. To obtain further confirmation of the expected product ion structure, MS3 was then performed on the m/z 178 product ion (Figures 3.5C and D). As expected, the experimental and standard MS3 spectra were essentially identical. The most abundant product ions at m/z 161 and 150 are most likely due to the loss of NH3 and CO, respectively. Based on an MS4 experiment (data not shown), the MS3 product ion seen at m/z 179 was formed via an ion-molecule reaction between the ion at m/z 161 and a water molecule. Taken together, the MS/MS and MS3 spectra provided strong evidence that the product seen at m/z 196 from the reaction of DHNP with SaDHNA is HP. This same type of analysis was also performed in order to determine the identity of the m/z 182 ion in Figure 5B. MS/MS and MS3 spectra of the ion were obtained and compared to those of the proposed reaction product, DHXP. An examination of Figure 3.6 indicated that the major ion observed by CID MS/MS of the ion at m/z 182 obtained from the SaY54F-catalyzed reaction is identical to the major ion seen in the MS/MS product ion spectrum obtained from the standard (compare Figures 3.6A and B). The m/z 154 product ion in Figures 3.6A and B is most likely due to the loss of CO from the precursor ion. MS3 was then performed on the m/z 154 ion (Figures 3.6C and D). Again, the experimental and standard spectra were essentially the same. The major MS3 product ion, seen at m/z 126, corresponds to the loss of CO, while the second most intense ion, seen at m/z 137, corresponds to the loss of NH3. As was the case for the previous analysis of the SaDHNA-catalyzed reaction, these spectra together provided strong 93 Figure 3.6: Multistage tandem mass spectrometry identification of the m/z 182 product from the reaction of DHNP with SaY54F. (A) CID MS/MS product ion spectrum of the ion at m/z 182 obtained from Figure 3.48. (B) CID MS/MS product ion spectrum of the m/z 182 precursor ion obtained from a solution of commercially available DHXP. (C) CID MS3 product ion spectrum of the ion at m/z 154 in panel A. (D) CID MS3 product ion spectrum of the ion at m/z 154 in panel B. 94 mg n): CNN CCN Cw CCC CE CN_ CC_ CC CC CNN CCN CM: CCC CE CN_ CC~ CC CC C LL _ p p _ _ _ _ ._ _L._ . _ _ _ FFL _ _ _ L ._ _ _jr11_ . _ T .. m__.N__ m__.N__ 1 Vm_ hN— ww_ hN_ .8953: - .8953; - hm_ hm— M:2. n=2. CN— CN— 8- Q -2: 8- o .2: CNN CCN Cm CC_ CE CN_ CC~ Cw CC CNN CCN C2 CC~ C3 CN~ CC_ Cw CC FF _ _ 1. p r F F _ _ FL f_ _ r _ p _ _ ._ _ 1|_| FL _ _ . _ _ _ _ _ Nw_ Nw_ .E+E 1 .E+Z - - T ww_ VC— 00. m— rCC_ 8. < -CCH aouepunqv QAIIEPH % aouepunqv 91111121921 % 95 evidence that the product seen at m/z 182 from the reaction of DHNP with SaY54F is DHXP. The m/z 256 ion (Figure 3.4) was found to correspond to DHNP (Figure 3.7), as well as probably partly due to DHMP, which was generated by the enzymatic reactions. DHNP and DHMP differ only in the stereochemistry of 2’-carbon and have the same molecular weight. The m/z 194 ion (Figure 3.4) was found to correspond to HPO and FDHP, because the MS/MS and MS3 spectra of the m/z 194 species were the sum of those of HPO and FDHP standards, with HPO contributing more than FDHP (Figure 3.8). The existence of FDHP was also confirmed by spectrophotometry, in which FDHP has a characteristic absorption peak at 420 nm (data not shown). GC/MS Confirmation of GA. Since the major product of the SaY54F-catalyzed reaction was DHXP, not HP, it was necessary to confirm the identity of GA as indicated by the NMR analysis (Figure 3.3). To this end, we derivatized the reaction products of SaDHNA and SaY54F with methoxyamine and MTBSTFA to form the tert- butyldimethylsilyl ether of the methoxime. GA was then detected by GC/MS as shown in Figure 3.9. The enzymatic reaction product (Figure 3.93) was identified by comparison with the commercially available standard GA (Figure 3.9A). The retention time of GA standard is 12.3 min (data not shown). The molecular weight of the GA derivative is 203 Da, but the molecular ion was too weak to be observed as is typical for these derivatives. The dominant fragment ions in the mass spectrum appeared at m/z 146 ([M-C4H9]+) and 188 ([M-CH3]+). Both reaction mixtures generated by SaDHNA and SaY54F contained a peak with the same retention time as that of standard GA. The mass spectrum of the 96 Figure 3.7: Multistage tandem mass spectrometry identification of the substrate DHNP. (A) CID MS/MS product ion spectrum of the ion at m/z 256 obtained from the reaction of DHNP with SaDHNA at 0 min. (B) CID MS/MS product ion spectrum of the m/z 256 precursor ion obtained from a solution of commercially available DHNP. (C) CID MS3 product ion spectrum of the ion at m/z 154 in panel A. (D) CID MS3 product ion spectrum of the ion at m/z 154 in panel B. 97 NE CwN CVN CCN CC~ CNC Cw ___._____P.T%k_jp_p P. C: 1 NC— me no. .Hoamiifi - W CNN CCN o”:. Q .2: CCN CVN CCN CC_ CN Cw — _ _ p _ . r r _ _ _ L _F p F p p p P . q. q _NN CC— CmN .Eia mom - me of- m CC— NE CwN CVN CCN of CN_ Cw — p w _ _ . _ . _ _ _ T r. 1_ _ _ _ _ b _ _ a: - «mm ..ofiifi - NE a: omN - o m- as U .2: CwN CVN CCN CC_ CNC Cw _ L p p _ p p L _ r _ p _ _ _ _ _ . _ _ p p _ T a 1 a; we mom - e2 .53. - wmm of- < -CC_ ooueptmqv analog % souepunqv SAQBIQH % 98 Figure 3.8: Multistage tandem mass spectrometry identification of the m/z 194 products from the DHNP reaction catalyzed by SaY54F. (A) CID MS/MS product ion spectrum of the ion at m/z 194 obtained from the Figure 3.43. (B) CID MS/MS product ion spectrum of the m/z 194 precursor ion obtained from a solution of commercially available HPO. (C) CID MS/MS product ion spectrum of the m/z 194 precursor ion obtained from a solution of commercially available 6-formyl-7,8- dihydropterin (F DHP). (D) CID MS3 product ion spectrum of the ion at m/z 176 in panel A. (E) CID MS3 product ion spectrum of the ion at m/z 176 in panel B. (F) CID MS3 product ion spectrum of the ion at m/z 165 in panel A. (G) CID MS3 product ion spectrum of the ion at m/z 165 in panel C. 99 NE. NE CNN C2 C3 CC_ CC CNN C2 C3 CC~ CC _r___r.L r. "_FCLF _____._ T___._ \ g 4 2. m2 \ o2 . a: \ \ o2 . Aamnm a . t + £2 om— +H¢Nnm+a w¢~ Om.— n=2. . £2- . t; _ . E . U of- N2 . 2: m of- N2 . 2: NE CNN Cw_ C3 CC_ CC CNN C2 C3 CC“ CC _ _ p k _. _ _ fir— P _ _ _ j. _ p r _ _ _ F p L H b . _ p _ _ — _ _ m: _42 s w: 42 . e: oo- o: co ..ofiifi . .Ho~:-:+§ . m— ? .CS D we -CCC CNN CM: CE CCC CC CNN of C3 CC~ CC CNN Cw _ Cg CC _ CC prr_j__r_._.L_rp_ rr_d____.__%k_pbh_ rp_w_.__~._k____r f 1 . we . X: r 2.: a: . .E+E . firs: . .212. . U m o: < 8: m2 . C2 of- . CC_ of- . CC_ SOUBpUHQV GAIIBIQH % GOUBPUIIQV GARBPH % aouepunqv analog % 100 Figure 3.7: Electron ionization mass spectra of the tert— butyldimethylsilyl/methoxime derivatives of GA standard (A) and the DHNP reaction mixture generated by SaY54F (B). The chromatographic retention times of the derivatives were 12.3 min. 101 500000 ' 450000 ‘ 400000 ‘ 350000 ‘ (a) O O O O 0 250000 ‘ Abundance ‘ 200000 ‘ 150000 1 1 00000 1 500001 0 . H3CO _N 73 100 H3CO_N \ ea CH3 H o_31_c\CH / 1 \ 3 O—Sl® CH 3CH3 \ CH 188 116 130 0 60 70 80 90 1(1) 11012)1301£K)1501601'X)l&)19) 2(1) 210 4800000. 4600000- 4400000. 42000001 4000000. 3800000. 3600000' 34000001 32000001 3000000. 2800000. 26000001 2400000. _2200000‘ 20000001 18000001 16000001 1400000. 1200000. 1000000. 800000‘ 6000001 4000001 2000001 Abundance 0 vi! V—V vvvvvvvvvvvvvvvv 116 130 146 188 0 60 70 80 90 1(1) 1101111301401501601701m190 200 210 102 m/z derivative (the peak at 12.3 min) from the SaY54F-catalyzed reaction (Figure 3.9B), which was identical to that of the SaDHNA-catalyzed reaction (data not shown), also showed two dominant ions at m/z 146 and 188. This result and that of the NMR analysis clearly indicated that the SaY54F-catalyzed reaction generates GA as the SaDHNA- catalyzed reaction. HPLC Identification of Formic Acid. Because DHXP contains one carbon less than HP, the SaY54F-catalyzed reaction must generate a one-carbon species, most likely formic acid. In order to identify the one-carbon species, we analyzed the DHNP reaction mixtures generated by SaDHNA and SaY54F by HPLC using an Aminex HPX-87H column. The unknown compounds in the reaction mixtures were identified by comparing their retention times with those of the standard compounds as shown in Figure 3.10. This HPLC run of standard compounds (blue line) included succinic acid at 13.5 min, formic acid at 14.9 min, and acetic acid at 16 min. It also showed a peak at 17.5 min, probably due to the HEPES buffer, which also appeared in all chromatograms, including those of the SaY54F solution (green line), the reaction mixture generated by SaY54F (red line), the reaction mixture generated by SaDHNA (cyan line), and the buffer alone (data not shown). The chromatogram of the reaction mixture generated by SaY54F showed an intense peak at 14.9 min, which was the same as the retention time for standard formic acid. The small shoulder at the left side was probably due to the protein. The intense peak in the chromatogram of the reaction mixture generated by SaY54F was absent in the chromatogram of the reaction mixture generated by SaDHNA. The result indicated that the mutant-catalyzed reaction generates formic acid but the wild-type enzyme-catalyzed reaction does not. 103 Figure 3.10: Identification of formic acid by HPLC. The red, cyan, green, and blue lines are the chromatogram of the SaY54F-catalyzed DHNP reaction mixture, the SaDHNA-catalyzed DHNP reaction mixture, the SaY54F solution, and a standard organic acid solution, respectively. The standard organic acid solution contained formic acid, acetic acid, and succinic acid. 104 0.006 _ Formic . Succinic acud 0.005 - 3°” 0.004 - 8 ‘ Acetic g 0.003 - acid .0 . 3 m 0.002 - _g Buffer < ‘ Protein 0.001 - A __ 0.000 - J \‘ e K -0.001 . . . , . , 14 16 1 8 Time (min) 105 Is DHXP Derived fiom HP? Since the C1’-C2’ bond is cleaved first in the generation of DXHP, which is the same for the normal aldolase reaction for the formation of HP, it is logical to ask whether DHXP is derived from HP. To address this question, we analyzed the time course of the DHNP reaction by MS. The results are shown in Figure 3.11. The ions at m/z 256, 196, and 182 were due to DHNP, HP, and DHXP, respectively, as described earlier. The m/z 194 ions were to a large part from HPO and to a small part from FDHP. The results showed again that the major product of the wild- type enzyme-catalyzed reaction was HP as expected, but the major product of the mutant- catalyzed reaction was DHXP. Furthermore, there was no accumulation of HP in the mutant-catalyzed reaction, suggesting that DHXP was not deriVed from HP, unless the conversion of HP to DHXP was much faster than the formation of HP. In order to confirm that DHXP was not formed from the rapid conversion of HP, HP was incubated with SaY54F and analyzed by MS. If DHXP was derived from the rapid conversion of HP, then over time, these spectra should show a decrease in the intensity of m/z 196 and an increase in the intensity of m/z 182. There was not, however, any production of the ion at m/z 182 seen in these spectra (not shown), indicating that DXHP was not derived from HP. The Source of Oxygen. Finally we considered the source of the new oxygen at the C-6 position of DHXP. Was it from water or from the oxygen molecules dissolved in the buffer? To address this issue, we ran the DHNP reaction in buffer prepared with '80- water and analyzed the reaction mixture with MS. With l8O-water, if the oxygen was from the solvent, the mass spectrum should show a shift in the m/z of the protonated 106 Figure 3.11: ESI-MS time course analysis of the DHNP reactions catalyzed by SaDHNA (A) and SaY54F (B). (o = m/z 256, u = m/z 194, A = m/z 196, o = m/z 182) 107 36:85 a2 3883.. Rama—85 c2 36:85 :2 CoEEsm 3:285 :3 10 Reaction Time (min) 108 product ion from 182 to 184. The MS spectra of the reaction mixtures obtained with 180- water as the solvent, however, were the same as the spectra obtained when the reaction was run in unlabelled solvent (data not shown), indicating that there was no '80 incorporation in any of the products and the oxygen incorporated in the product DHXP does not come from water. Then we measured the oxygen consumption of the reactions catalyzed by SaY54F and SaDHNA. The results are shown in Figure 3.12. The amounts of wild-type and mutant enzymes were both 8 1.1M. Under the conditions, nearly all DHNP was converted to products. The results showed that only the mutant-catalyzed reaction consumes a significant amount of oxygen, indicating that SaY54F is an oxygenase and the source of oxygen for the oxygenation reaction is molecular oxygen dissolved in the buffer. We calculated the turnover number for the oxygenation reaction from the linear portion of the oxygen consumption curve, which was 0.027 s". We also calculated the turnover number for the formation of DHXP from the NMR time course of the SaY54F-catalyzed reaction, which was 0.018 s'l. The two turnover numbers were in close agreement, indicating that one oxygen molecule is used in the formation of one molecule of DHXP. The extra oxygen consumption was probably due to the formation of FDHP and HPO. DISCUSSION SaY54 and EcY53F Are Oxygenases. By a variety of means we have shown that the major product of the mutant (either SaY54F or EcY53F)-catalyzed reaction is DHXP, rather than HP of the wild-type enzyme-catalyzed reaction. The turnover number for the 109 Figure 3.12: Oxygen consumption by the reactions catalyzed by SaDHNA and SaY54F. The initial samples contained 200 uM DHNP in 100 mM HEPES-KOH, pH 8.2, and the reactions were initiated by 8 uM SaDHNA or SaY54F as marked by the vertical lines. 110 Oxygen (11M) 250 _ SaDHNA 200- SaY54F 150- 100- J 50- 0 400 800 1200 1600 Time (s) 111 formation of DHXP is >50-fold that for the formation of the normal product HP for SaY54F. The initial clue to the surprising properties of the mutant enzymes was a dramatic drop of the fluorescence intensities of the reaction mixtures. The NMR analysis of the reaction mixtures revealed that the mutant enzymes generate a new compound with a 1H NMR signal at 4.12 ppm and little of the normal product HP (Figure 3.3). The new compound was also a major product in the MS analysis of the reaction mixtures generated by the mutant SaY54F (Figure 3.4) and was identified to be DHXP by comparing its MS/MS and MS3 spectra with those of a DHXP standard (Figure 3.6). This raises the immediate question of how DHXP is generated. Is DHXP generated via the cleavage of the C6-Cl’ bond or the C1 ’-C2’ bond as in the aldolase reaction? The fact that either mutant-catalyzed reaction generates GA and furthermore that about equal moles of GA and DHXP are generated (Figure 3.3) strongly indicates that DHXP is generated via the cleavage of the Cl’-C2’ bond. The formation of GA was confirmed by the derivatization of the reaction mixtures followed by GC-MS analysis (Figure 3.9). Since the first step in the generation of DHXP is the cleavage of the C1’-C2’ bond as in the wild-type enzyme-catalyzed reaction, the next question is whether DHXP is derived from HP. The MS analysis of the time course of the mutant-catalyzed reaction shows that there is no accumulation of HP in the reaction (Figure 3.12). This indicates that DHXP is not derived from HP, unless the conversion of HP to DHXP is very rapid relative to the generation of HP. This was addressed by mixing HP standard with the mutant and following the possible reaction by MS. The result indicates that HP cannot be converted to DHXP under the experimental conditions. Since the cleavage of the Cl’-C2’ bond generates an intermediate with one carbon more than DHXP, the conversion of the 112 intermediate to DHXP must generate a one-carbon species, which was identified as formic acid by comparing the HPLC chromatogram of the reaction mixtures generated by the mutant and wild-type enzymes with those of standard organic acids and aldehydes (Figure 3.10). The remaining question is where the oxygen at the C-6 position of DHXP comes from. Is it from water or from oxygen molecules dissolved in the buffer? This issue was addressed by the D's-water experiment and the oxygen consumption assay. The Q's-water experiment eliminates water as the source for the oxygen newly attached to the pterin ring. The oxygen consumption assay indicates that molecular oxygen is the source for the oxygenation reaction, because significant oxygen consumption only occurs in the mutant catalyzed reaction (Figure 3.11) and the turnover number calculated from the oxygen consumption data is comparable to that for the formation of DHXP calculated from the NMR data. The above analysis leads us to propose a chemical mechanism for the mutant- catalyzed reaction as depicted in Figure 3.13. The initial step in the formation of DHXP is the generation of the same enol intermediate as in the wild-type enzyme-catalyzed reaction, which involves the cleavage of the Cl’-C2’ bond and yields approximately equal moles of GA and DHXP, as described earlier. Deprotonation of N5 of the enol intermediate generates a carbanion species, which donates a single electron to molecular oxygen to form a caged radical pair. The caged radicals react to generate the peroxide ion, which leads to the formation of DHXP and formic acid. The participation of molecular oxygen in the mutant-catalyzed reaction has been confirmed by the oxygen consumption assay. The reaction path for the formation of DHXP from the enol intermediate is speculative, but the formation of formic acid has been confirmed by 113 HPLC. The proposed mechanism also accounts for the formation of the minor product FDHP. Alternative mechanisms involve protein radicals (in contrast to just substrate radicals). Both mechanisms have been proposed for cofactor—independent oxygenases (10), but the proposed mechanism or variations thereof that involve mainly general acid and base catalysis are more likely, particularly considering that DHNA is evolved for general acid and base catalysis. The key step in the formation of DHXP from the enol intermediate is the generation of the carbanion species, which involves the deprotonation of N5 as in the wild-type enzyme-catalyzed reaction and the block of the protonation of the hydroxymethylene group by the mutation of the active site tyrosine residue. Carbanions are prone to react with oxygen, and several enzymatic reactions involving carbanion intermediates indeed have oxygen-consuming side reactions (11, 12). The formation of the peroxide ion is reminiscent of the initial steps of the reactions catalyzed by 1H-3- hydroxy-4-oxoquinaldine 2,4-dioxygenase (13) and urate oxidase (14, 15), both of which do not have any cofactor. These enzymes seem to evolve to utilize carbanion reactivity toward dioxygen for the oxygenation reactions (10), which might be a general catalytic strategy for the family of cofactor-independent oxygenases. Interestingly, both DHNA (4, 7, 16, 17) and urate oxidase (18, 19) have a tunnel-like structure called “T-fold” with active sites located between subunits (20). The bound ligands are stacked with a conserved phenylalanine residue in urate oxidase and the conserved tyrosine in DHNA that was substituted with a phenylalanine residue in this study. The Role of the Conserved Tyrosine Residue. The biochemical analysis of the two mutants SaY54F and EcY53F indicates that the hydroxyl group of the phenol ring of the 114 conserved tyrosine residue plays only a minor role in the physical steps of the enzymatic reaction. In contrast, the hydroxyl group plays a critical role in catalysis. The hallmark of the DHNA-catalyzed reaction is general acid and base catalysis. The general acid‘and base involved in the protonation and deprotonation of N5 are most likely a water molecule and its conjugated base based on the published crystal structures (7) and our own unpublished crystal structures. The deprotonation of 2’-hydroxyl group is most likely the function of the conserved lysine residue, K100 in SaDHNA and K98 in EcDHNA. The conserved tyrosine residue may play a minor role if any in this step, because the formation of the enol intermediate is not impaired by the mutations to any great extent as evidenced by the high km for the formation of DHXP by the mutant SaY54F, which is 1/10 of the kw value for the formation of HP by the wild-type enzyme. On the other hand, the major product of the mutant-catalyzed reaction is DHXP, not HP, suggesting that the protonation of the enol intermediate to generate HP is greatly impaired and the conserved tyrosine residue may play a critical role in this step. The high oxygenase activities of the mutants are likely due to the tendency of the enol intermediate to react with molecular oxygen and the available general acid and base for the generation of the enol intermediate and its subsequent oxygenation as illustrated in Figure 3.13. The conserved tyrosine residue is located at the bottom of the active site. The active site is likely to be accessible to molecular oxygen even in the wild-type enzyme. The wild-type enzyme is an efficient aldolase rather than an oxygenase, because it has a general acid that can efficiently protonate the enol intermediate to form HP. 115 Figure 3.13: The proposed chemical mechanism for the generation of DHXP and FDHP by SaY54F and EcY53F. For simplicity, many steps are indicated by single arrows irrespective of their reversibility. 116 _ -H 0 OH 0 9H 3 20 : HN \ 1' ' OH HN \ v OH 131' SW A ' I? HzN C HzN N N a . :B DHNP '3 DHMP A-“V Az’V o 171 OH 0 iii,) OH N / N / o HN HN ' C» t / r T Afi )6, ” H _ \ H B H2NJ\\N N H B OGAOH H2N N n 1. OH 02 HN N\ OH O\ \ l .B + ‘\ H2N N u ° 0“ oA-H OH N —/\ HP HN I \ 00—0. \ HZN N N _ H A: H o *1 | \f+q formlcacld o — \ oA—H (0 0 HzN N N/ CH 117 REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) Walsh, C. (2003) Where will new antibiotics come from? Nature Rev. Microbiol. 1, 65—70. Bermingham, A., and Derrick, J. P. (2002) The folic acid biosynthesis pathway in bacteria: Evaluation of potential for antibacterial drug discovery. Bioessays 24, 637-648. Kompis, I. M., Islam, K., and Then, R. L. (2005) DNA and RNA synthesis: Antifolates. Chem. Rev. 105, 593-620. Sanders, W. J ., Nienaber, V. L., Lerner, C. G., McCall, J. O., Merrick, S. M., Swanson, S. J ., Harlan, J. E., Stoll, V. S., Stamper, G. F., Betz, S. F., Condroski, K. R., Meadows, R. P., Severin, J. M., Walter, K. A., Magdalinos, P., Jakob, C. G., Wagner, R., and Beutel, B. A. (2004) Discovery of potent inhibitors of dihydroneopterin aldolase using crystalead high-throughput X-ray crystallographic screening and structure-directed lead optimization. .1. Med. Chem. 47, 1709-1718. Mathis, J. B., and Brown, G. M. (1970) The biosynthesis of folic acid XI. Purification and properties of dihydroneopterin aldolase. J. Biol. Chem. 245 , 3015-3025. HauBmann, C., Rohdich, F., Schmidt, E., Bacher, A., and Richter, F. (1998) Biosynthesis of pteridines in Escherichia coli - structural and mechanistic similarity of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase. J. Biol. Chem. 273, 17418-17424. Hennig, M., D'Arcy, A., Hampele, I. C., Page, M. G. P., Oefner, C., and Dale, G. E. (1998) Crystal structure and reaction mechanism of 7,8- dihydroneopterin aldolase from Staphylococcus aureus. Nature Struct. Biol. 5, 357-362. Scherperel, G., Yan, H. G., Wang, Y., and Reid, G. E. (2006) 'top-down' characterization of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase by multistage tandem mass spectrometry in a linear quadrupole ion trap. Analyst 131, 291-302. Li, Y., Gong, Y., Shi, G., Blaszczyk, J., Ji, X., and Yan, H. (2002) Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6- hydroxymethyl-7,8-dihydropterin pyrophosphokinase. Biochemistry 41, 8777- 8783. Fetzner, S. (2002) Oxygenases without requirement for cofactors or metal ions. Appl. Microbiol. Biotechnol. 60, 243-257. 118 (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) Abell, L. M., and Schloss, J. V. (1991) Oxygenase side reactions of acetolactate synthase and other carbanion-forming enzymes. Biochemistry 30, 7883-7887. Hixon, M., Sinerius, G., Schneider, A., Walter, C., Fessner, W. D., and Schloss, J. V. (1996) Quo vadis photorespiration: A tale of two aldolases. FEBS Lett. 392, 281-284. Frerichs-Deeken, U., Ranguelova, K., Kappl, R., Huttennann, J ., and F etzner, S. (2004) Dioxygenases without requirement for cofactors and their chemical model reaction: Compulsory order ternary complex mechanism of 1H-3-hydroxy-4- oxoquinaldine 2,4—dioxygenase involving general base catalysis by histidine 251 and single-electron oxidation of the substrate dianion. Biochemistry 43, 14485- 14499. Sarma, A. D., and Tipton, P. A. (2000) Evidence for urate hydroperoxide as an intermediate in the urate oxidase reaction. J. Am. Chem. Soc. 122, 11252-11253. Imhoff, R. D., Power, N. P., Borrok, M. J ., and Tipton, P. A. (2003) General base catalysis in the urate oxidase reaction: Evidence for a novel Thr-Lys catalytic diad. Biochemistry 42, 4094-4100. ‘ Bauer, 8., Schott, A. K., Illarionova, V., Bacher, A., Huber, R., and Fischer, M. (2004) Biosynthesis of tetrahydrofolate in plants: Crystal structure of 7,8- dihydroneopterin aldolase from Arabidopsis thaliana reveals a novel adolase class. J. Mol. Biol. 339, 967-979. Goulding, C. W., Apostol, M. I., Sawaya, M. R., Phillips, M., Parseghian, A., and Eisenberg, D. (2005) Regulation by oligomerization in a mycobacterial folate biosynthetic enzyme. J. Mol. Biol. 349, 61-72. Colloc'h, N., ElHajji, M., Bachet, B., Lherrnite, G., Schiltz, M., Prange, T., Castro, B., and Momon, J. P. (1997) Crystal structure of the protein drug urate oxidase-inhibitor complex at 2.05 angstrom resolution. Nat. Struct. Biol. 4, 947- 952. Retailleau, P., Colloc'h, N., Vivares, D., Bonnete, F., Castro, 8., El Hajji, M., Momon, J. P., Monard, G., and Prange, T. (2004) Complexed and ligand-free high-resolution structures of urate oxidase (on) from aspergillus flavus: A reassignment of the active-site binding. Acta Crystallogr. D. Biol. Crystallogr. 60, 453-462. Colloc'h, N., Poupon, A., and Momon, J. P. (2000) Sequence and structural features of the T-fold, an original tunnelling building unit. Proteins 39, 142-154. 119 CHAPTER 4: THE FUNCTIONAL ROLES OF THE CONSERVED ACTIVE SITE GLUTAMATE AND LYSINE RESIDUES ABSTRACT Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8- dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 in Staphylococcus aureus DHNA (SaDHNA), corresponding to E21, Y53, E73, and [(98 in Escherichia coli DHNA (EcDHNA). Previously we have shown that the conserved tyrosine residue plays a critical role 'in the protonation of the enol reaction intermediate to form HP. The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and 521, E73, and K98 of EcDHNA were replaced by alanine. K100 of SaDHNA was replaced by alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. The results showed that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. In contrast, I522 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis. 120 INTRODUTION Dihydroneopterin aldolase (DHNA) catalyzes the conversion of the 7,8- dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway, one of principal targets for developing antimicrobial agents (1). Folate cofactors are essential for life (2). Most microorganisms must synthesize folates de novo. In contrast, mammals cannot synthesize folates because of the lack of three enzymes in the middle of the folate pathway and obtain folates from the diet. DHNA is the first of the three enzymes that are absent in mammals and therefore an attractive target for developing antimicrobial agents (3). DHNA is a unique aldolase in two respects. First, DHNA requires neither the formation of a Schiff base between the substrate and enzyme nor metal ions for catalysis (4). Aldolases can be divided into two classes based on their catalytic mechanisms (5, 6). Class I aldolases require the formation of a Schiff base between an amino group of the enzyme and the carbonyl of the substrate, whereas class II aldolases require a Zn2+ ion at their active sites for catalysis. The proposed catalytic mechanism for DHNA is similar to that of class I aldolases, but the Schiff base is embedded in the substrate. Secondly, in addition to the aldolase reaction, DHNA also catalyzes the epimerization at the 2’-carbon of DHNP to generate 7 ,8-dihydromonapterin (DHMP) (Figure 1.3) (7), but the biological function of the epimerase reaction is not known at present. Interestingly, DHNAs from Gram-positive and Gram-negative bacteria have some unique sequence motifs. Figure 1.2 shows the amino acid sequence alignment of DHNAs from 11 bacteria. The first five enzymes are from Gram-positive bacteria, and the rest are 121 F1... from Gram-negative bacteria. The identities between enzymes from Gram-positive bacteria range from 39% to 45% and those between Gram-negative bacteria are 49-91%, but the identities between Gram-positive and Gram-negative bacterial enzymes are <30%. Many differences between these enzymes from Gram-positive and Gram-negative bacteria are at or near their active centers (8). We have recently shown that DHNAs from Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) indeed have significant differences in their binding and catalytic properties (Wang et al., unpublished), suggesting that it is possible to develop antimicrobial agents targeting a specific class of bacteria. DHNA consists of eight identical subunits. The atomic structures of SaDHNA (3, 8), Mycobacterium tuberculosis DHNA (MtDHNA) (9), and Arabidopsis thaliana DHNA (AtDHNA) (10) have been determined by X-ray crystallography. The octameric structures look like two stacked donuts with a large hole in the middle, ~13 A in SaDHNA. Each donut consists of four subunits. There are eight active sites, all formed by residues from two adjacent subunits. At the active sites, there are four conserved residues that interact with the bound product HP as revealed by the crystal structures (8, 9) (Figure 1.7). These four residues are 522, Y54, E74, and K100 in SaDHNA, corresponding to E21, Y53, E73, and K98 in EcDHNA, respectively (Figure 1.2). Previously we showed that the conserved tyrosine residue plays a critical role in DHNA catalysis. Substitution of the conserved tyrosine residue in SaDHNA or EcDHNA with phenylalanine turned the target enzyme into an oxygenase. In this paper, we describe a site-directed mutagenesis study of the functional roles of the other conserved, active-site residues in SaDHNA and EcDHNA. The results provide important insight into the 122 catalytic mechanisms of the enzymes and valuable information for designing inhibitors targeting these enzymes. EXPERIMENTAL PROCEDURES Materials. 6-Hydroxymethylpterin (HPO), 6-Hydroxymethyl-7,8-dihydropterin (HP), 7,8-dihydro-D-neopterin (DHNP), 7,8-dihydro-L-monapterin (DHMP), D- neopterin (NP), and L-monapterin (MP) were purchased from Schircks Laboratories. Pfu DNA polymerase was purchased from Strategene. Other chemicals were from Sigma or Aldrich. Site-Directed Mutagenesis and Protein Purification. The site-directed mutants were made by a PCR-based method using high-fidelity pfu DNA polymerase according to a protocol developed by Stratagene. The forward and reverse primers for the PCR-based mutagenesis experiments are listed in Table 4.1. The mutants were selected by DNA sequencing. In order to ensure that there were no unintended mutations in the mutants, the entire coding sequences of the mutated genes were determined. The mutant proteins were purified as described previously (Wang et al., unpublished). Briefly, SaE22A, SaE74A, SaK100A, and SaK100Q were purified to homogeneity by a Ni-NTA column followed by a Bio-Gel A-0.5m gel column. EcE21A, EcE73A, and EcK98A were purified by a DEAE-cellulose column followed by a Bio-Gel A-0.5m gel column. The purities of the protein preparations were checked by SDS- PAGE. The purified proteins were concentrated, dialyzed, lyophilized, and stored at -80 °C. 123 Table 4.1: The Forward and Reverse Primers for the PCR-Based Mutagenesis Experiments aSaE22A, SaE74A, and SaK100A are mutants of SaDHNA in which E22, E74, and K100 are replaced by alanine respectively. SaKlOOQ is a SaDHNA mutant with K100 replaced by glutamine. EcE21A, EcE73A, and EcK98A are mutants of EcDHNA in which E21, E73, and K98 are replaced by alanine respectively. bThe forward primers are listed first with the mutations underlined. The mutations in the reverse primers are not indicated. 124 ((3111:: and 311? Mutantal Primerb SaE22A 5 ’ -GGTGCTTTATCAGCTGQAAATGAAATAGGGCAAATTTTC-3 ’ 5 ’ -GAAAATTTGCCCTATTTCATTTGCAGCTGATAAAGCACC-3 SaE 74A 5 ’ -GCCGTTAATTTACTTGQGCATCTAGCTGAACGTATTGC-3 ’ 5 ’ -GCAATACGTTCAGCTAGATGCGCAAGTAAATTAACGGC-3 ’ SaK 100A 5 ’ - GAAACGAAAGTGAGAATCACTQQAGAAAACCCACCGATTCCG-3 ’ 5 ’ -CGGAATCGGTGGGTTTTCTGCAGTGATTCTCACTTTCGTTTC- 3 9 SaK 1 COO 5 ’ -CGAAAGTGAGAATCACTQAAGAAAACCCACCGATTCC-3 ’ 5’- GGAATCGGTGGGTTTTCTTGAGTGATTCTCACTTTC G -3’ EcE2 1 A 5 ’-GTGTTTACGACTGGGQACAGACCATCGAACAG-3 ’ 5 ’-CTGTTCGATGGTCTGTGCCCAGTCGTAAACAC-3 ’ EcE73A 5 ’-GCGCTGGTGGQACGCGTGGCTGB ’ 5’-CAGCCACGCGTGCCACCAGCGC-3 ’ EcK98A 5’-CGTATCAAACTCAGCQQGCCAGGCGCAGTGGJ ’ 5 ’-CCACTGCGCCTGGCGCGCTGAGTTTGATACG-3 ’ 125 Equilibrium Binding Studies. The procedures for the equilibrium binding studies of DHNAs were essentially the same as previously described for the similar studies of 6- hydroxymethyl-7,8-dihydropterin pyrophosphokinase using a Spex F luoroMax-2 fluorometer (11, 12). Briefly, proteins and ligands were all dissolved in 100 mM Tris- HCl, pH 8.3 and the titration experiments were performed in a single cuvette at 24 oC. The equilibrium binding experiments were performed by titrating either ligands or the proteins. In ligand titrations, fluorescence intensities were measured at an emission wavelength of 446 nm with a slit of 5 nm. The excitation wavelength and slit were 400 nm and 1 nm, respectively. A set of control data was obtained in the absence of the protein. The data set obtained in the absence of the protein was then subtracted from the corresponding data set obtained in the presence of the protein after correcting inner filter effects. The K., value was obtained by nonlinear least square fitting of the titration data as previously described (11). All Kd values for SaDHNA mutants except that of SaE22A for HPO were obtained by titrating ligands. In protein titration experiments, a ligand solution was titrated with a stock solution of one of the mutant proteins. Fluorescence intensities were measured at an emission wavelength of 430 nm and an excitation wavelength of 330 nm. The emission and excitation slits were both 5 nm. A control titration experiment was performed in the absence of the ligand. The control data set obtained in the absence of HP was subtracted from the corresponding data set obtained in the presence of the ligand. The Kd value was obtained by nonlinear least square fitting of the titration data as previously described (11). All Kd values for EcDHNA mutants and that of SaE22A for HPO were obtained by titrating proteins. 126 Stopped-F low Analysis. Stopped-flow experiments were performed on an Applied Photophysics SX.18MV-R stopped-flow spectrofluorometer at 25 °C. One syringe contained one of the mutant proteins, and the other contained NP, MP, HP or HPO. The protein concentrations were 1 or 2 uM, and the ligand concentrations ranged from 5-60 uM. All concentrations were those after the mixing of the two syringe solutions. Fluorescence traces for NP, MP and HPO were obtained with an excitation wavelength of 360 nm and a filter with a cutoff of 395 nm for emission. Fluorescence traces for HP were obtained with an excitation wavelength of 330 nm and the same filter for emission. Apparent rate constants were obtained by nonlinear squares fitting of the data to a single exponential equation and were re-plotted against the ligand concentrations. The association and dissociation rate constants were obtained by linear regression of the apparent rate constants vs. ligand concentration data. Steady-State Kinetic Assay. All components were dissolved in a buffer containing 100 mM Tris-HCl, 1 mM EDTA, and 5 mM DTT, pH 8.3. The reactions were initiated by mixing with the mutant enzymes and quenched with 1 N HCl. The quenched reaction mixtures were processed as previously described (7). Briefly, the reaction mixtures (115 pl each) were mixed with 50 pl 1% 12 (w/v) and 2% (w/v) K1 in 1 N HCl for 5 min at room temperature to oxidize the pterin compounds. Excess iodine was reduced by mixing with 25 ul 2% ascorbic acid (w/v). The samples were then centrifuged at room temperature for 5 min using a microcentrifuge. The oxidized reactant and products in the supematants were separated by HPLC using a Vydac RP18 column. The column was equilibrated with 20 mM NaHzPO4 made with MilliQ water and eluted at the flow rate of 0.8 ml/min with the same solution. The oxidized reactant and products were quantified by 127 online fluorometry with excitation and emission wavelengths of 365 and 446 nm, respectively. The enzyme and substrate concentrations were adjusted appropriately on the basis of initial estimations of the kinetic constants. When the substrate concentrations were in large excess of the enzyme, the normal Michaelis-Menten equation was used to obtain kinetic constants by nonlinear least square regression. When the enzyme concentration is comparable to the substrate concentrations because of the extremely low activity of the enzyme, a modified steady state kinetic equation (Equation 1) was used to obtained kinetic constants. v=53L1(E,+s,+Km-J(E,+S,+Km)2—4E,s,) (1) where E, and S. are the total enzyme and substrate concentrations, respectively, km and Km have the usual meanings. RESULTS Binding Studies. Previously we established the thermodynamic and kinetic framework for the structure-function studies of SaDHNA and EcDHNA by equilibrium measurements and stopped-flow and quench-flow analyses (Wang et al., unpublished). A similar strategy was used for the characterization of the site-directed mutants. The binding steps were mimicked with the substrate and product analogues NP, MP, and HPO. The only difference between the substrate and product analogues and the corresponding substrate and products is that between C7 and N8 is a double bond in the analogues and a single bond in the substrate and products. NP, MP, and HPO are excellent substrate and product analogues, as described previously. The Kd values were 128 igure 4.1: Binding of MP to SaE22A at equilibrium. A 2 mL solution containing 10 uM SaE22A in 100 mM Tris-HCl, pH 8.3, was titrated with MP by adding aliquots of a 1.03 mM MP stock solution at 24 °C. The final enzyme concentration was 9.3 M. The top axis indicates the MP concentrations during the titration. A set of control data was obtained in the absence of the enzyme and was subtracted from the corresponding data set obtained in the presence of the enzyme. The solid line was obtained by nonlinear least-squares regression as previously described (I I). 129 (m <5 sofa The IT - —l q —1 - d C: 120000- 1000001 80000: 60000: 40000- Fluorescence 20000d 0- -20000 I V I U I V I T r -20 o 20 40 60 80 100 120 140 160 MP(HL) 130 160 Figure 4.2: Binding of RFC to EcK98A at equilibrium. A 2 mL solution containing 5 uM HPO in 100 mM Tris-HCl, pH 8.3, was titrated with EcK98A by adding aliquots of a 1.9 mM EcK98A stock solution at 24 °C. The final HPO concentration was 4.6 uM. The top axis indicates the EcK98A concentrations during the titration. A set of control data was obtained in the absence of HPO and was subtracted from the corresponding data set obtained in the presence of HPO. The solid line was obtained by nonlinear least—squares regression as previously described (11). 131 Fluorescence 0 19 37 55 72 90 106 123 139 (..M) 180000- 160000: 140000: 120000: 100000: 80000: 60000; 40000~ 1 20000- I I I ' I I ‘ I I -20 r 0 I 20"401'60 '8b .100 1 EcK98A (uL) 132 I j I 20 14 . , . 0 160 180 measured by equilibrium binding experiments using fluorometry by either titrating the ligands or the proteins. Representative results of the equilibrium binding studies are shown in Figures 4.1 and 4.2. The association and dissociation rate constants were determined by kinetic binding experiments using st0pped-flow fluorometry. A representative result of the kinetic binding studies is shown in Figures 4.3. For technical reasons, the binding constants for SaE74A and the association and dissociation rate constants for EcE73A could not be measured. The complete results of the binding studies are summarized in Tables 4.2 and 4.3 for SaDHNA and EcDHNA, respectively. In general, the thermodynamic data are in good agreement with the kinetic data, as the measured Kd values are in good agreement with those calculated from the association and dissociation rate constants. High Kd values are, for the most part, due to high dissociation rate constants. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. The Kd values for SaE74A were estimated to be >3000 uM, suggesting that the Kd values of the mutant is at least 100 times those of the wild-type enzyme. The results indicated that for SaDHNA, of the three conserved residues, only E74 is important for the binding of the analogues. For EcDHNA, similarly, the mutation of E73, corresponding E74 in SaDHNA, had the most dramatic effects on the Iigand binding. The E73A mutation caused increases in the Kd values for the binding of NP, MP and HPO by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme, suggesting that E73 is critically important for the binding of these substrate or product analogues. In addition, the K98A mutation caused increases in the Kd values for the binding of NP, MP, and HPO by factors of 14, 3.6, and 230, respectively, suggesting that K98 is important for 133 Figure 4.3: Stopped-flow fluorometric analysis of the binding of HPO to SaE22A. The concentration of SaE22A was 1 11M, and the concentrations of HPO were 5, 10, 20 and 40 uM for traces 1, 2, 3, and 4, respectively. All concentrations were those immediately after the mixing of the two syringe solutions. Both SaE22A and HPO were dissolved in 100 mM 100 mM Tris-HCl, pH 8.3. The fluorescent signals were rescaled so that they could be fitted into the figure with clarity. The solid lines were obtained by nonlinear regression as described in the Experimental Procedures section. Panel B is a replot of the apparent rate constants vs. the HPO concentrations. The solid line was obtained by linear regression. 134 0.340- 0.3353 0.3301 0.3253 0.320- 0.315; 0.310j 13“ 0.305; 0.300- Fluorescence LA AL- . AA A A "WV wwwvv—v' "ww 32 31:1. so 304 28 - 241 22? 20- 181 161 144 121 kapp (3-1) 10 .1 1O 15 ' 20 . 25 MP (“W 135 30 35 40 45 ‘ ..M“ Table 4.2: Binding Constants of SaDHNA and Site-Directed Mutantsa SaDHNA SaE22A SaE74A SaK100A SaKlOOQ NP K., (.071) 18:1:2 13i0.6 >4000 6.9i0.7 11420.3 k1(HM"s") 0.24i0.01 0.29i0.008 n.d. 0.2240004 0.1940007 k.) (5") 45:0,] 4.03:0.1 n.d. 1.22.007 2.03.008 MP K. (HM) 1321 11i0.9 >3000 9.1i0.6 133:1 k1 (11M"s") 0.293.002 0.31.2001 n.d. 0.27:t0.006 0.28:1:0.002 k.1 (8") 4.2202 40.0.1 n.d. 2.1i0.l 4.3:t0.1 HPO Kd(uM) 2410.2 1742 >6000 60:60.1 8.8i0.4 k1 (11M"s“) 0.452.002 0.56:1:0.03 n.d. 0.64d:0.03 0683:004 k.] (5") 10105 10:02 n.d. 5.8:t0.3 5.8:t0.1 3Both the wild-type SaDHNA and mutants have a His-tag at the N-terminus. We have shown previously that the His-tag has no effects on the binding and catalytic properties of the enzyme. The chemical structures of the measured compounds are as follows. 0 “NW A / H2N N N 0 OH N N NP HZN 0 MP 136 9H N ’ N ' HN/UI W014 HNJEE j/YOH )x\ / 0H )\\ / 01" H2N N N HPO Table 4.3: Binding Constants of EcDHNA and Site-Directed Mutants EcDHNA EcE21A EcE73A EcK98A NP K., (1.1M) O.77:l:0.06 1.7-.6001 2604:40 1 13:03 k1 (uM'ls'l) O.32i0.02 0.46i0.02 n.d.a 0.66d:0.007 k.1 (s'l) 0.29:1:0.03 0.822t0.02 n.d. 7,210.3 MP K. (0M) 2.6:t0.06 0.80:1:0.01 420.220 9.42.009 k1 (uM’ls'l) 0.26:1:0.0l 0433:002 n.d. 0.911004 k.) (s") 0.583003 0.472002 n.d. 9.13:0.6 HPO K. (M) 0103:0007 4.25:0.01 560:1:20 23i2 kl (uM'ls'l 0.553.004 1.4:.004 n.d. 12320.03 k-1 (s'l) 0.062i0.006 6.3:t0.2 n.d. 2m 8|n.d.: not determined. 137 the binding of these ligands, particularly for the binding of HPO and NP. The E21A mutation caused increases in the Kd values for the binding of NP and HP by factors of 2.2 and 42, respectively, but a decrease in the Kd value for the binding of MP by a factor of 3.3, suggesting that E21 is only important for the binding of HPO. Steady-State Kinetic Studies. The catalytic properties of the mutants were determined by steady-state kinetic measurements. Because the reactions were slow, no quench-flow apparatus was needed. For technical reasons, mainly because of the solubility limits of DHNP and DHMP, only kmt/Km could be estimated for SaF74A. Also, only kw could be estimated for SaKlOOQ, because the fluorescence of some unknown small molecules associated with the protein preparation caused significant errors in the reaction rates at low substrate concentrations. The steady-state kinetic parameters of the SaDHNA and EcDHNA mutants are summarized in Tables 4.4 and 4.5, respectively. Probably because of the complicated kinetic mechanism (Figure 2.1), there appears to be no correlation between the Km values measured by the steady-state kinetic experiments and the corresponding Kd values measured by the binding studies. Thus, our analysis of the steady-state kinetic data focuses on the kw values of the mutants. For SaDHNA, the E22A mutation caused a decrease in kcatby a factor of 4.8><103 with DHNP as the substrate and 1.5><103 with DHMP as the substrate, in comparison with those of the wild- type enzyme. The K100A and KIOOQ mutations caused decreases in kc... by factors of 2><104 and 2.8X103, respectively, with DHNP as the substrate, and by factors of 2><103 and 1.8X103, respectively, with DHMP as the substrate. The effects of the two mutations were very similar. The results suggest that both E22 and K100 are important for catalysis. The kcat value of SaE74A mutant could not be determined, but its kcm/Km value could be 138 Table 4.4: Steady State Kinetic Constants of SaDHNA and Site-Directed Mutants SaDHNA SaE22A SaE74A SaKl 00A SaK l OOQ DHNP 16.0") Km (11M) kcat/Km (8"1LM") DHMP k... (s") Km (HM) kcat/Km (8"uM") 004520002 (0320.4)x10'6 n.d.a (2.22010106 (1.6:0.3)><10‘5 4.6:t0.3 3.9:t0.6 n.d. 5.83:1.1 n.d. 9.7><10'3 2.4><10'6 (1.7:t0.09)><10'5 3.7x10'7 0.0120001 (6.5i0.2)><10'6 n.d. (5.1:t0.1)><10'6 (5.7;1:0.9)><10‘6 5.5:t0.2 4.0i0.6 n.d. 9.5e07 n.d. 1.8X10‘3 1.6XI0’6 (8.03:0.08)><10'5 5.4><10'7 an.d.: not determined. 139 Table 4.5: Steady State Kinetic Constants of EcDHNA and Site-Directed Mutants EcDHNA EcE21A EcE73A EcK98A DHNP k...(s") 008230.001 (6.5:t0.3)><10'5 (7.3:t0.6)><10'3 (4.3i003)x10'6 K.,, (1.1M) 7.4a 0.3 1.6i 0.3 9700i1000 2.4301 k,,./Km(s"pM") 1.1><10'2 4.2><10'5 7.6><10'7 1.8x10'6 DHMP k.,.(s") 0089320004 (2.33.0.2)x10" (6.0:tl)><10'3 (5.320.05)><10'6 K.,. (0M) 8.0:t0.6 0.76:t0.2 9800:8000 2.9i0.2 heat/K.,,(s'luM'l) 1.1><10‘2 3.1x10'5 6.l><10‘7 1.8><10"S 140 estimated from the linear part of the reaction rate vs. substrate concentration curve, which decreased by a factor of 570 with DHNP as the substrate and a factor of 23 with DHMP as the substrate. The decreases in the hug/Km values are probably largely due to the increases in Km, considering that the mutation caused dramatic decreases in the affinities of the enzyme for all substrate or product analogues. The result suggests that E74 plays no great role in catalysis. This was confirmed by the mutation of the corresponding residue (E73) of EcDHNA. The E73A mutation of EcDHNA caused a decrease in kc... by only a factor of ~10. The EcE21A and EcK98A mutants behaved like the corresponding SaDHNA mutants (SaE22A and SaK100A) in terms of their kw values. Thus, the km of EcE21A decreased by a factor of 1.3><103 with DHNP as the substrate and by a factor of 3.9><103 with DHMP as the substrate, in comparison with those of the wild-type E. coli enzyme. The kg,“ of EcK98A decreased by a factor of l.9><104 with DHNP as the substrate and by a factor of 1.7><104 with DHMP as the substrate. The results suggest that the conserved glutamate and lysine residues both are important as well for the catalysis by EcDHNA. DISCUSSION Figure 2.1 illustrates the proposed chemical mechanism for the DHNA-catalyzed reaction. It is understood that the enzymatic reaction takes place in the confines of the eight active sites of the octomeric enzyme. For simplicity, the physical steps of substrate binding and product dissociation are omitted. The epimerization reaction is proposed to occur via the intermediate for the retroaldol reaction, which is supported by our NMR 141 analysis of the reaction in D20 that shows no deuteration of 2’H of DHMP (Wang et al., unpublished). However, little is known about how DHNA catalyzes the reaction. Of the published crystal structures DHNAs (3, 8-10), the most informative structures are the binary HP complexes of SaDHNA and MtDHNA, which reveal the atomic interactions between the pterin moiety of the substrate and the enzymes. Four conserved residues and an important water molecule are found at the active site as illustrated in Figure 1.7 for SaDHNA. The structure of SaDHNA in complex with the substrate analogue NP (3) should provide the structural information about the interaction between the trihydroxypropyl moiety and the enzyme. Unfortunately, the occupancy value is 0 with an R factor of 100 for all trihydroxypropyl atoms of the bound NP, suggesting that the trihydroxypropyl moiety of NP was not ‘seen in the crystal and no structural information about the interaction between the trihydroxypropyl moiety and the enzyme can be deduced from the crystal structure. The interactions between pterin and DHNA are reminiscent of those of dihydrofolate reductase (DHF R) with dihydrofolate, which also contains a pterin moiety. The common features include two hydrogen bonds between a carboxylate group of a glutamate or aspartate and the 2-NH2 and 3-N H groups of the pterin and a hydrogen bond between a water molecule and N5 of the pterin. Replacement of D27 of E. coli DHFR, a residue corresponding to E74 of SaDHNA and E73 of EcDHNA, with asparagine or serine causes a significant decrease in km and a significant increase in Km or K., suggesting that the aspartate is important for both substrate binding and catalysis (13). On the other hand, replacement of D26 of Lactobacillus casei DHF R, which corresponds to D27 of E. coli DHFR, with asparagine causes a <10-fold decrease in km and essentially 142 no change in Km or Kd, suggesting that the carboxyl group is not important for substrate binding but may play a minor role in catalysis (14). For DHNA, the biochemical properties of SaE74A and EcE73A indicate that the conserved glutamate is very important for substrate binding and its role in catalysis is a minor one if any. It contributes to the binding of the pterin compounds by 3-5 kcal/mol on the basis of the binding data of the two mutants. The hallmark of the DHNA-catalyzed reaction is general acid and base catalysis (Figure 2.1). The formation of the intermediate of the retroaldol reaction requires the protonation of N5 and the deprotonation of 2’-OH of DHNP. The formation of HP requires the deprotonation of S-NH and the protonation of the enol group of the reaction intermediate. The epimerization reaction is basically the reversal of the chemical step for the formation of the reaction intermediate following the flip of GA. We have recently shown that the conserved active site tyrosine residue, corresponding to Y54 in SaDHNA and Y53 in EcDHNA, plays a critical role in the protonation of the enol group of the reaction intermediate to form HP (Wang et al., unpublished). Replacement of either Y54 of SaDHNA or Y53 of EcDHNA causes a dramatic decrease in the rate for the formation of HP but no significant change in the rate for the formation of the reaction intermediate. Either mutation converts the aldolase to an oxygenase. The water molecule that is hydrogen bonded to N5 of HP in the crystal structures (8, 9) is probably the general acid for the protonation of N5 of DHNP and its conjugated base for the deprotonation of S-NH of the reaction intermediate, because no amino acid residue is in a position to play such a role according to the crystal structures. There are several candidate residues that may act as a general base for the deprotonation of 2’-OH of DHNP according to the crystal 143 structures, including E22, Y54, and K100 (SaDHNA numbering). Y54 can be excluded on the basis of our previous site-directed mutagenesis of the conserved tyrosine residue (Wang et al., unpublished). The present site-directed mutagenesis study suggests that both E22 and K100 are important for catalysis, with K100 contributing a bit more to the transition state stabilization. The larger contribution by K100 is probably due to its hydrogen bond with the water molecule that serves as a general acid in the first chemical step of the enzymatic reaction. However, from the chemical perspective, K100 is much more likely to serve as a general base for the deprotonation of 2’-OH of DHNP. The optimal pH for the DHNA-catalyzed reaction is 9.6 (4, 15). In the crystal structure of the product complex of SaDHNA, K100 is hydrogen bonded to the carboxyl group of E22, the hydroxyl group of Y54, and the water molecule that serves as a general acid. It probably has a normal pK8| of ~10, which matches closely with the optimal pH of the enzymatic reaction. On the other hand, the carboxyl group of E22 is hydrogen bonded to the hydroxyl of HP, the main-chain NH of L19, and the side-chain amide of Q27. The pKa of E22 is unlikely to be higher than 4.5, the pKa of model peptides, which is too far away from the optimal pH of the enzymatic reaction. While E74 of SaDHNA and the corresponding residue E73 in EcDHNA play a common role in the enzymatic reaction, namely in the binding of the substrate, the roles of E22 and K100 of SaDHNA are slightly different from those corresponding residues E21 and K98 in EcDHNA. Both E22 and K100 of SaDHNA are involved in catalysis but neither contributes to the binding of the substrate. On the other hand, in addition to their roles in catalysis, both E21 and K98 are also involved in the binding of the substrate. The different biochemical properties between SaDHNA and EcDHNA revealed by the 144 previous study of the wild-type enzymes and this site-directed mutagenesis study suggest that it is possible to develop specific inhibitors for these two enzymes. 145 REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) Walsh, C. (2003) Where will new antibiotics come from? Nature Rev. Microbiol. 1, 65-70. Blakley, R. L., and Benkovic, S. J. (1984) in Folates and pterins, John Wiley & Sons, New York. Sanders, W. J., Nienaber, V. L., Lerner, C. G., McCall, J. O., Merrick, S. M., Swanson, S. J., Harlan, J. E., Stoll, V. S., Stamper, G. F., Betz, S. F ., Condroski, K. R., Meadows, R. P., Severin, J. M., Walter, K. A., Magdalinos, P., Jakob, C. G., Wagner, R., and Beutel, B. A. (2004) Discovery of potent inhibitors of dihydroneopterin aldolase using crystalead high-throughput X-ray crystallographic screening and structure-directed lead optimization. J. Med. Chem. 47, 1709-1718. Mathis, J. B., and Brown, G. M. (1970) The biosynthesis of folic acid XI. Purification and properties of dihydroneopterin aldolase. J. Biol. Chem. 245 , 3015-3025. , Horecker, B. L., Tsolas, O., and Lai, C.-Y. (1975) Aldolases, in The enzymes (Boyer, P. D., Ed.) pp 213-258, Academic Press, San Diego. Allen, K. N. (1998) Reactions of enzyme-derived enamines, in Comprehensive biological catalysis (Sinnott, M., Ed.) pp 135-172, Academic Press, San Diego. HauBmann, C., Rohdich, F ., Schmidt, E., Bacher, A., and Richter, F. (1998) Biosynthesis of pteridines in Escherichia coli - structural and mechanistic similarity of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase. J. Biol. Chem. 273, 17418-17424. Hennig, M., D'Arcy, A., Hampele, I. C., Page, M. G. P., Oefner, C., and Dale, G. E. (1998) Crystal structure and reaction mechanism of 7 ,8- dihydroneopterin aldolase from Staphylococcus aureus. Nature Struct. Biol. 5, 357-362. Goulding, C. W., Apostol, M. I., Sawaya, M. R., Phillips, M., Parseghian, A., and Eisenberg, D. (2005) Regulation by oligomerization in a mycobacterial folate biosynthetic enzyme. J. Mol. Biol. 349, 61-72. Bauer, 8., Schott, A. K., Illarionova, V., Bacher, A., Huber, R., and Fischer, M. (2004) Biosynthesis of tetrahydrofolate in plants: Crystal structure of 7,8- dihydroneopterin aldolase from arabidopsis thaliana reveals a novel adolase class. J. Mol. Biol. 339, 967-979. 146 (11) (12) (13) (14) (15) Li, Y., Gong, Y., Shi, G., Blaszczyk, J ., Ji, X., and Yan, H. (2002) Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6- hydroxymethyl-7,8-dihydropterin pyrophosphokinase. Biochemistry 41, 8777- 8783. Shi, G., Gong, Y., Savchenko, A., Zeikus, J. G., Xiao, B., Ji, X., and Yan, H. (2000) Dissecting the nucleotide binding properties of Escherichia coli 6- hydroxymethyl-7,8-dihydropterin pyrophosphokinase with fluorescent 3'(2)'-o- anthraniloyladenosine 5'-triphosphate. Biochim. Biophys. Acta 1 4 78, 289-299. Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J ., and Kraut, J. (1986) Functional role of aspartic acid-27 in dihydrofolate-reductase revealed by mutagenesis. Science 231, 1 123-1128. Birdsall, B., Casarotto, M. G., Cheung, H. T. A., Basran, J ., Roberts, G. C. K., and Feeney, J. (1997) The influence of aspartate 26 on the tautomeric forms of folate bound to lactobacillus casei dihydrofolate reductase. FEBS Lett. 402, 157- 161. Mathis, J. B., and Brown, G. M. (1980) Dihydroneopterin aldolase from Escherichia coli. Methods Enzymol. 66, 556-560. 147 MICHIGAN SIATE UNIVERSITV LIBRARIES ill/illlllllll/lllllllllllllill/ll 3 1293 02845 0652