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MECHANISTIC EVALUATION AND SUBSTRATE SPECIFICITY
ANALYSIS OF A PHENYLALANINE AMINOMUTASE

By

Washington Mutatu

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements for
the degree of
DOCTOR OF PHILISOPHY
Chemistry

2010

ABSTRACT

MECHANISTIC EVALUATION AND SUBSTRATE SPECIFICITY
ANALYSIS OF A PHENYLALANINE AMINOMUTASE

By
Washington Mutatu
Phenylalanine aminomutase (PAM) derived from Taxus is a
multifunctional enzyme that catalyzes the reversible vicinal exchange of the
amino group and the pro-(3S) hydrogen of various (28)-a-arylalanines to make

the corresponding (3Ff)-B-arylalanines, with trans-cinnamic acid as a major by-
product. A combination of 1H- and 2H-NMFI experiments were employed to
analyze the stereochemistry at 02 of the B-phenylalanine product catalyzed by
PAM. [3,3-2H21-(2S)-a-phenylalanine was incubated with PAM, and the
biosyntheticaIIy-derived 2H-labeled B-phenylalanine derivatized as the N-acetyl
methyl ester, revealed deuterium chemical shift values consistent with those of
[2,3-2H21-(28,3R)-and (2R,3S)-B-phenylalanine racemate. Considering that chiral
GC-MS analysis confirmed the B-product to be the (3Ff)-enantiomer, and that an
earlier study showed that the amino group migrates with retention of
configuration at its terminus, the mutase was therefore shown to

stereospecifically shuttle the pro-(38) hydrogen to C2 with retention of

configuration.

The deuterium kinetic isotope effect on Vmax/KM is 2.0 :I: 0.2, indicating

that CB-H bond cleavage is rate limiting. The migratory deuterium of [2H3]-(23)-CI-

phenylalanine was shown to dynamically exchange (up to 70 %) with

protons from the solvent during the isomerization. The equilibrium constant of the
enzyme for the natural substrate is 1.1 at 30 °C.

The PAM reaction does not require an external cofactors but involves a
post transcriptionally and autocatalytically synthesized 3,5
-dihydro-5-methylidene-4H-imidazolone (MIO) moiety which is believed to assist
in shuttling the amino group between theaandBpositions of the substrate. The
presence of the catalytic motif in PAM was confirmed by mutagenesis. Mutants
$176A, 81766 and S176T were used as blanks against wild-type enzyme in
difference UV-visible spectroscopy with monitoring of the signature peak at 308
nm as observed in similar experiments with tyrosine aminomutase that also
contain an MIO. The presence of the MIC in PAM was further confirmed by
inhibition with cysteine at pH 10.5 under both aerobic and anaerobic conditions.
An absorbance peak at 338 nm was observed which is typical of the formation of
a cysteine-MIC complex as seen for the ammonia lyases. This absorbance
maximum was absent when PAM mutants S176A, G and T were analyzed
analogously. The PAM mutants (S176A, G T) converted 4’-nitro— and 4'-
flourophenylalanines to their B-isomers. Likely, the MIC, at least in part functions
to reduce the pKa of the Bhydrogens to facilitate the removal of the pro-38
hydrogen by a general base within the active site.

When E. coli transinfected with a plasmid carrying the pam gene is grown
under light illumination it has a higher level of endogenously synthesized B

phenylalanine compared to non-illuminating conditions.

 

I dedicate this work to my wife Jane, my daughter Vongaishe, and sons, Trinity,
Rangariro, and Farai. I hope they will be inspired to work hard in all their

endeavors.

ACKNOWLEDGEMENTS

I would like to acknowledge my advisor Dr. Kevin. D. Walker, for his
guidance and patience during the course of my research. Without his direction
and advice, I would not have had much progress. Dr. Joan Broderick who is now
at Montana State University deserves special mention. She was very
instrumental in getting me to the USA and enrolling me into the graduate
program. When I joined her group in 2003, I learnt many biotechnical skills, which
became valuable to the successful of my project. Many thanks goes to the
following people who were all part of the Walker group; lrosha for encouraging
me to attend relevant seminars and for assisting me with the protein sequence
alignment computer program. I am also deeply thankful of Karin for introducing
me to the PAM project and training me how to operate the GC-MS instrument; to
Mark, Getrude, Koyeli, Danielle for editing my thesis; Benhaz , Yemane, Brad
Cox, Sean, Joshua, Uday for their helpful criticism. Moreover, I am grateful for
Getrude and Koyeli for continuing the project to further dissect the mechanism of
the PAM reaction. I am also thankful to my second reader, committee member
Dr. James Tiedje, for his advice and encouragement and to the rest of my
guidance committee: Dr. James McCusker, Dr James Geiger and Dr. Flawle
Hollingsworth who acted as both a mentor and, as well as a role model and
provided inspiration. Also recognized are Dr. Daniel Holmes and Mr. Kermit

Johnson of the Max T. Rogers NMFI Facility at Michigan State University for their

assistance in acquiring the 2H-, 13C-, 15N- NMR spectra. Dr. Daniel Jones for

run the QToff mass spectrometer and analyze the results. Thanks also goes
Doug Whitten for carrying out PAM peptide sequencing; the results were useful
in confirming phosphorylation of the enzyme. I would also like to acknowledge
Dr. Mitch Smith for allowing working in his lab, wherein I acquire skills to
synthesized small molecules and running NMR experiments.

Many thanks goes to my family: Jane for being supportive and checking
on my progress regularly, Trinity for setting a good example to his siblings,
Flangariro for his desire to pursue Engineering, Farai for the energy and
sportsmanship, and Vongai for keeping me busy whenever I was not working on
my research. I also thank my friend Gasseller and his wife Pauline for
encouraging me to play tennis, which kept me energized. Dr. Edward
Mazhangara and his family assisted me in settling in East Lansing during the
initial days of my program, and for this, I am most thankful. Pastor Dave Williams
of Mt. Hope church provided me with spiritual encouragement, which made my

life more meaningful.

vi

PREFACE

Chapter 1 covers a detailed investigation on the stereochemistry of the
migratory hydrogen on the CZ position of the Beta product. Chapter 2 includes an
evaluation of the substrate specificity of PAM and the discussion on the effects of
electronic and inductive effects on the reactivity of the substrate. Chapter 3
covers the general characterization of PAM, in terms of the kinetics, growth,
overexpression and purification optimization and general assay techniques.
. Chapter 3 also covers mutagenetic investigation of the role of the MIO cofactor
on the activity and functionality of PAM and an investigation into the role of a
general base during the PAM catalysis is also described. Chapter 4 is the
investigation of the effect of light on the activity of PAM as measured by the

amount of endogenous B phenylalanine. Chapter 5 was an attempt to establish
the mechanism of the reaction by utilizing 130 and 15N NMR and analyzed the

intermediates and transient states formed during the reaction.

vii

TABLE OF CONTENTS

LIST OF TABLES ............................................... xi
LIST OF FIGURES ............................................. xii
LIST OF SCHEMES ............................................ xx
LIST OF ABBREVIATIONS ...................................... xxii
CHAPTER 1

ASSESSMENT OF THE CRYPTIC STEREOCHEMISTRY OF
PHENYLALANINE AMINOMUTASE: RETENTION OF CONFIGURATION AT

THE MIGRATION TERMINI ....................................... 1
1.1 Introduction ........................................... 1
1.2 Results ............................................... 8
1.2.1 Hydrogen Exchange at C2 of the PAM Reaction Product ...... 8
1.2.2 Complete Stereochemistry of the PAM Reaction ............ 11
1.2.3 The Rate Determining Step on the Isomerization Pathway. . . .18
1.2.4 Equilibrium Constant Measurements ...................... 19
1.3 Discussion ........................................... 20
1.3.1 Hydrogen Washout during Isomerization Chemistry .......... 20
1.3.2 Stereochemical Fate of the PAM Reaction ................. 25
1.3.3 Substrate/Product Distribution at Equilibrium ............... 29
1.4 Conclusions .......................................... 30
1.5 Materials and Methods ................................. 32
1.6 References ........................................... 45

CHAPTER 2

ANALYSIS OF THE SUBSTRATE SPECIFICITY OF PHENYLALANINE

AMINOMUTASE (PAM) ......................................... 56
2.1 Introduction ......................................... 56
2.2 Results and Discussion ................................ 65
2.2.2.1 Substrate Specificity Evaluation ....................... 65
2.2.2.2 Stereochemistry of the B-amino acids .................... 67
2.2.2.3 In vivo Screening of Amino acids ...................... 71
2.2.2.4 Biocatalysis ....................................... 72
2.2.2.5 Mass Spectral Analysis of Biosynthetic Products Generated

by PAM Catalysis .................................. 75

2.3 Conclusions and Recommendations ...................... 88
2.4 Materials and Methods ................................ 89
2.4.1 Materials and Reagents .............................. 89

2.4.2 Procedures ......................................... 91

2.4.2.1 Heterologous Expression and Purification of PAM ......... 91
2.4.2.2 Assessing Functional PAM Expression .................. 92
2.4.2.3 Screening of productive substrates ..................... 92
2.4.2.4 Quantification of BiosyntheticaIIy-derived B-Amino Acids. . . 94
2.4.2.5 Determining the Relative Kinetic Constants for

various "Allylglycine" Substrates ....................... 95
2.5 References .......................................... 103

CHAPTER 3
CHARACTERIZATION OF PHENYLALANINE AMINOMUTASE: CONFIRMING

THE PRESENCE AND ROLE OF 3,5-DIHYDRO-5-METHYLIDENE-4-H-

lMIDAZOL-4-ONE (MIO) ....................................... 117
3.1Introduction.................. ....................... 117
3.2 Results and Discussion ................................ 129
3.2.1 Optimization of the over expression of phenylalanine
aminomutase ...................................... 129
3.2.2 Enzymatic Assay Techniques .......................... 130
3.2.3 The PAM pH Dependence ............................. 131
3.2.4 Cysteine Inhibition Studies ............................ 132
3.2.5 Expression and Purification of Recombinant Wild-type PAM
and Mutants S176A, 8176C, S176G and Sl76T ......... 134
3.2.2 Discussion ......................................... 142
3.3 Conclusions ........................................ 151
3.4 Materials and Methods ................................ 153
3.5 References .......................................... 163
CHAPTER 4

RECOMBINANT EXPRESSED PHENYLALANINE AMINOMUTASE (PAM)
SHOWS INCREASED ACTIVITY in E. coll GROWN UNDER LIGHT

ILLUMINATION ............................................... 173
4.1 Introduction ......................................... 173
4.2 Results and Discussion ............................... 179
4.2.1 Results ............................................ 179
4.2.1.1 Assaying for PAM activity ............................ 179
4.2.1.2 Effect of lysis technique on PAM activity measurement

under light illumination conditions .................... 180
4.2.1.3 Investigation of the effect of UV light on the activity of

pure PAM ........................................ 181
4.2.1.4 Effect of sonication and light illumination on pure PAM. . . . 182
4.2.1.4 Effect of pre-sonication on the activity of pure PAM ........ 183
4.2.1.6 Time Course Assay ................................ 185
4.2.1.7.1 Monitoring of E. Coli Growth under Light Illumination

Conditions ..................................... 185

4.2.1.7.2 Effect of Different Types of Radiation on the Activity

of Recombinant PAM Grown in E. Coli ................ 186

4.2.1.8 Identifying Phosporylation Sites ...................... 187

4.2.2 Discussion ......................................... 188

4.3 Conclusions ......................................... 193

4.4 Materials and Methods ................................ 194

4.5 References ........................................... 201
CHAPTER 5

AN ATTEMPT TO DECIPHER THE MECHANISM OF PHENYLALANINE
AMINOMUTASE (PAM) USING AN ISOTOPICALLY ENRICHED

SUBSTRATE ................................................. 218
5.1 Introduction ......................................... 218
5.2 Results and Discussion ............................... 226
5.2.1.1 13C NMR Spectrum of the Substrate ................... 226
5.2.1.2 Kinetic Array of the PAM Reaction Monitored by ‘30 NMR. .228
5.2.1.3 Identification of Products of the PAM Reaction ............ 229
5.2.1.4 Identification of Intermediates ......................... 232
5.2.1.5 15N NMR Spectra .................................. 235
5.2.1.6 Relative Kinetic Rates of the Substrate, trans-cinnamic

Acid and B-phenylalanine formation .................... 237
5.2.2 Discussion ......................................... 238
5.3 Conclusions ........................................ 242
5.4 Materials And Methods ................................ 244
5.5 References .......................................... 247

LIST OF TABLES

Table 1.1 A summary of the H/D exchange during the PAM reaction. Diagnostic
ions resulting from electron impact-mass spectrometry fragmentation. Shown are
mass spectral fragments after bond fissure at sites 'a' and 'b', yielding diagnostic
ions of the corresponding N-acetyl-d-phenylalanine methyl ester ............ 10

Table 1.2 Summary of the source, substrates, products, cofactor requirement
and the stereochemistry of the products of various aminomutases ........... 28

Table 2.1 Kinetic Parameters of PAM with various substrates. The Vrel is the ratio
of Vmax for a particular substrate and Vmax of phenylalanine ............... 65

Table 3.1 Kinetics parameters of wild-type PAM with substrates L-phenylalanine,
4'-nitro-L-phenylalanine, 4'-fluoro-L-phenylalanine and 4'-methoxy-L-
phenylalanine ................................................... 137

Table 3.2 Comparison of kinetic parameters from wild—type PAM, the mutants
S176G, S176A, S176T and S176C with 4-nitrophenylalanine as substrate. . . 178

Table 3.4 The primers used to construct mutants S176A, G, C and T, the
mutated codons are in bold underlined ............................... 158

Table 3.5 Primers used to sequence the pam gene ..................... 160

Table 3.6 The primers used to construct mutants Y322A,H and F, and 0107A
and H; the mutated codons are in bold underlined ...................... 162

xi

LIST OF FIGURES

Figure 1.1 The reaction of PAM with d-phenylalanine showing the retention of

configuration of the NH2 group migrating from C2 to C3 (A), confirming that only
the pro-(3R)-hydrogen migrates to the C2 position (B and C) ................ 5

Figure 1.2 The proposed mechanism of PAM involving the aromatic ring as
nucleophile (left) or the amine as nucleophile (right) ...................... 6

Figure 1.3 A partial sequence alignment of PAM_T. canadensis with other MIO
carrying enzymes PAM_T. chinensis, PAL_G. biloba, PAL_sylvestris, PAL_A.
thaliana, PAL_R. toruloides, TAM_S. globisporous, HAL_P. putida and TAL_R.
sphaeroides ...................................................... 6

Flgure1.4 Hydrogen exchange at the C2 position ........................ 9

Figure 1.5 1HNMR for a-phenylalanine (A), B-phenylalanine (B) and a mixture of
a- and B-phenylalanine (C), the amino acids were derivatized to their mixture of
N-acetylated methyl esters. The expanded regions showing the splitting patterns
are shown in the inserts below the full spectra .......................... 13

Figure 1.6 A. 2H-NMR for an enantiomeric mixture of [2,3-2H21-(2S,3FI)- and
(2R,3S)-B-phenylalanine as N-acetylated methyl esters. The peaks are observed

at 6 = 2.82 and 5.32 ppm for a- and B- ZD respectively. B. The 2H-NMR of [3,3-
2H2]-a-phenylalanine derivatized as the N—acetylated methyl ester ........... 14

Flgure 1.7 The Newman projects of diastereoisomers (2S,3R)-, (2R, 38)—[2-,3-
2H2]-N-acetyl-phenylalanine methyl ester .............................. 17

Figure 1.8 Study of the complete stereochemistry at the C2 position of the 8-
product with reference to the migratory Hydrogen from the C3 position ....... 18

Figure 1.9 Assessment of the equilibrium conditions of the PAM with respect to
the unreacted substrate and product. Dideteurated a-phenylalanine was
incubated with PAM at 30 °C until the reaction reached equilibrium (17 h) ..... 18

Figure 1.10 Measurement of the equilibrium constant of the PAM reaction with
the natural substrate. PAM (100 pg) was incubated with 100 pM substrate and
samples were at set time points and assayed as above. The o-substrate change
over time is shown in black dashes and the B-product in blue boxes ......... 19

Figure 1.11 The mechanism of PAL R. toluroides (steps vi and v)and the
proposed mechanism of phenylalanine aminomutase (PAM) (steps i-iii). Step (i)
is the proposed nucleophilic attack on the aromatic ring on the methylidene of the

MIC, followed by (ii) the collapse of the sigma complex and elimination of NH3 ,

xii

is the proposed nucleophilic attack on the aromatic ring on the methylidene of the
MIO, followed by (ii) the collapse of the sigma complex and elimination of NH3 ,
and step (iii) the rebound of the NH3 at the C3 position. Step (iv) is the sigma

complex formation and (v) is the NH3 elimination as proposed for the PAL
mechanism ...................................................... 21

Figure 1.12 Synthesis of (2S,3Fi)-, (2R,3S)-N-acetyl-j3-methyl ester
racemate ....................................................... 25

Figure 2.1 Structure of taxol® and Januvia ............................ 58

Figure 2.5 The aromatic ring attack on the terminal double of the catalytic MIO
could be blocked by the presence of a methyl group at the on‘ho- position of the
substrate L-phenylalanine leading to lower reactivity compared with the natural
substrate ....................................................... 69

Figure 2.6. Shows a model of phenylalanine anchored in the active site of PAM
with the phenyl ring interacting with hydrophobic residues. The approximate
interaction distances between the substrate and the active site residues are
indicated (A) ..................................................... 71

Figure 2.7 Shows the possible products of the a-allylglycine-PAM reaction. The
loss of the a-amino group could lead to an extended conjugated system which
may result in the addition on the v and 6 positions to form the 4- or 5- amino-
trans-pent-3-enoic acids. However both compounds were not identifiable by
GC/MS/EI analysis ..................................................
...... 72

Figure 2.8 A profile of invivo synthesis of a mixture of 8- amino acids. The a-
amino acids were incubated with E. coli transinfected with a pam plasmid
expressing phenylalanine aminomutase (PAM), at 37 °C overnight. The PAM

expression was induced with IPTG at an ODsoo ~ 0.6. The products were
assayed in both (C) cells and supernatant (S). 3M is 3'-methyI-B-phenylalanine;
4M is 4'—methyI-B-phenylalanine; 2M is 2'-methyl-B-phenylalanine; 3F is 3'-flouro-
B-phenylalanine; 4F is 4'-flouro-B-phenylalanine; 2F is 2'-flouro-B-phenylalanine;
Fu is 2'-furanyl-j3-alanine; Th is 2'-thienyl-B-alanine and 4MO is 4'-methoxy-B-
phenylalanine ...... 73

Figure 2.9 General scheme for the synthesis of various stereospecific beta
amino acids ..................................................... 74

Scheme 2.10 Biosynthesis of taxol from tit-phenylalanine which is first converted
to B-phenylalanine (boxed) by the enzyme phenylalanine aminomutase(PAM). 75

Figure 2.11 The proposed biocatalysis of of Taxol involving various enzyme
found in the biosynthetic pathway of taxol .............................. 76

xiii

Figure 2.12.1 N-benzoyl 4'-fluoro-B-phenylalanine methyl ester ............ 78

Figure 2.12.3 N-benzoyl 4'-methoxy-B-phenylalanine methyl ester .......... 79
Figure 2.12.2 N-benzoyl-B-(2'-thienyl)-B-alanine methyl ester .............. 80
Figure 2.12.4 N-benzoyl 2'-f|uoro-B-phenylalanine methyl ester ............. 81
Flgure 2.12.5 N-acetyl 4'-methyl-B-phenylalanine methyl ester ............. 82
Figure 2.12.6 N-acetyl B-phenylalanine methyl ester ..................... 83
Figure 2.12.7 N-acetyl 2'-methyl-B-phenylalanine methyl ester ............. 84
Figure 2.12.8 N-benzoyl 3'-fluoro-B-phenylalanine methyl ester ............ 85
Figure 2.12.9 N-acetyl 3'-methyI-B-phenylalanine methyl ester ............. 86
Figure 2.12.10 N-benzoyI-B-(Z-furanyl)-B-alanine methyl ester ............. 87
Figure 2.12.11 N-acetyl styryI-B-alanine methyl ester ..................... 88
Figure 2.12.12 N-acetyl 4'-t-butyI-B-phenylalanine methyl ester ............. 89
Figure 2.13 Some of the unnatural substrates tried with PAM .............. 95

Figure 3.2 shows the amine group of L-d-phenylalanine bound to. the MIO
prosthetic group. The pH of the B-hydrogen drops by nearly 40 pKa units. . . . 148

Figure 3.5 The mechanism of SgTAM based on crystal structure. The tyrosine
substrate is shown bound to the MIO via the amine group. Tyrosine 63 acts as a
general base to de-protonate the pro-(m-B-hydrogen stereospecifically. A
coumarate is formed as an intermediate. The MIO shuttles the amine group to
the 8 position, the subsequent breakdown of the MIO-amine complex results in
release of the beta amino acid as a product ........................... 126

Figure 3.6 The active sites of PAL R. tolouride at the top showing the binding of
the substrate at the active site ...................................... 127

Figure 3.7; SDS-PAGE of proteins expressed in BL21(DE3)-RIPL cells
transformed with a pam plasmid at 18 °C or 37 °C. The PAM protein with a size
of 76 kDa is barely distinct from the rest of the cellular proteins pointing to weak
expression. Lane 1 the Lonza protein marker with the 75 kDa bark shown on the
left. Lanes 2-9 are whole cells from different flasks and treated by spinning the

xiv

cell cultures at 13 000 rpm, boiling the pellet in BME for 20 min. Approximately
the same amount of protein was loaded in each lane .................... 129

Figure 3.8; SDS-PAGE of proteins expressed in BL21(DE3) RIPL cells
transformed with a pam plasmid at 16 oC. The PAM protein with a size of 76 kDa
(lanes 1,3,4,5 and 6) was over 60 % of the cellular proteins suggesting a
successful over expression. Lane 8 is the protein ladder (Lonza) and the 75 kDa
marker is indicated on the right ..................................... 130

Figure 3.9 The SDS-PAGE gel of elutions of PAM from the Ni-NTA column. The
PAM has a measured mass of about 75 :I: 5 kDa based on the protein ladder in
lane 1. The protein was over expressed in BL21 (DE3)CodonPlus-RIPL at 16 °C
and induced with 1 mM IPTG ....................................... 130

Figure 3.10 An activity assay PAM (50 pg) incubated for 3 hours. The 4'-
methoxyphenylalanine and 4'-Flourophenylalanine is converted to over 50 %, the
universally deuterated phenylalanine and the 4'-nitrophenylalanine are at 30 %
conversion ..................................................... 1 31

Figure 3.11 The pH dependence of the PAM reaction. The activity increases
rapidly form pH 6.5 and peaks at pH 8.5 before it gradually decreases at higher
pH ........................................................... 132

Figure 3.12 Spectra of the formation of the MIO-cysteine complex monitored
over a period of 60 min. The spectra were at 10 min intervals after incubating
with 10 mM L-cysteine ............................................ 133

Figure 3.13 The effect of cysteine inhibition on PAM activity. PAM was reacted
with 10 mM cysteine at 30 °C for 50 min. After 50 min the cysteine was removed
by filtering through a 30 kDa cut of Amicon filter and the activity of the enzyme
was assayed as described above and the activity of PAM was fully restored. .134

Figure 3.14 The SDS/PAGE gel with Coomassie staining showing over
expressed wild-type PAM mutant (lanes 1 and 3), S176A (lanes 4 and 7), S176G
(lanes 5 and 8), S176T (lanes 6 and 9), showing that the 4 proteins are of the
same size (75 :I: 5 kDa) ........................................... 168

Flgure 3.15 The difference spectra between the mutants SI76A/G/T versus wild-
type PAM exhibiting a Amax at 310 nm which increase in strength with increase
in the wild-type enzyme concentration ................................ 136

Figure 3.16 The activity of the mutant S176C was assayed by incubating with
various amino acids for a period of 3 h. The natural substrate showed more
reactivity than the 4’-F-Phe which exhibit higher with wild type PAM ........ 137

XV

Figure 3.17 The Lineweaver-Burk plot for wild-type PAM vs 4'-
nitrophenylalanine. The calculated KM value is 0.3 mM ................... 138

Figure 3.18 Plot of 1/Vmax versus 1/[S] for the reaction between the mutant
S176A and d-4'-nitrophenylalanine as substrate. The KM is 1 mM .......... 139

Figure 3.19 Activity of mutant Sf76G as measured by the amount mol°/c of
cinnamate formed from the a-amino acids (4'-nitrophenylalanine and 4'-
flourophenylalanine). The B-product was below the detection limit. The results
suggest the influence of the electron withdrawing groups on the phenyl ring of the
substrate ....................................................... 140

Figure 3.20 The Michael-Menten plot for mutant S176C with the 4'-
nitrophenylalanine substrate ........................................ 141

Figure 3.21 The assay to check the activity of the of the five mutants Y322A, F,
H and C107A, H was carried out by incubating the enzymes with 1 mM of the
natural substrate (4H) and 4'-flouro-o-phenylalanine (4F). Only mutants C107 A
and C107F showed activity ........................................ 142

Figure 3.23 The HPLC spectrum of a mixture of D and L phenylalanine. The
amino acids were separated with 100% methanol isocratic gradient and no
derivatization was necessary ....................................... 184

Figure 3.24 The nitro group withdraws electrons from the phenyl ring causing the
B-hydrogen to be acidic enough to be de-protonated by a general base. The
same electron withdrawing is proposed to apply with fluorinated substrates. . 186

Figure 3.26 The possible products formed from the reaction between crude wild-
type PAM with 4'-nitrophenylalanine. The reaction is complicated by the presence
of phenylalanine racemase form E. coli which isomerizes D phenylalanine to the
L isomer ....................................................... 150

Figure 3.27 Seven 0 helices associated with the active sites of PAL and HAL.
Shown are the six positive poles and one negative pole of the seven 0 helices,
directed towards active-site residues, with cofactor shown for reference ..... 151

Figure 4.1 A scheme showing the fate of L-phenylalanine in E. coli. The
productin of PAM in E. coli will increase the flux of trans-cinnamic acid thereby
upsetting the cellular metabolism which may contribute to different response to
light conditions .................................................. 179

Figure 4.2 Effect of total white light illumination on phenylalanine aminomutase
activity in E. coli at 16 °C. The percentage conversion of (S)-d-phenylalanine to

xvi

(FOB-phenylalanine in cell cultures exposed to total white light (left) and the
control kept in the dark (right). The amino acids were derivatized as described
above and assayed by GC/MS. The light illuminated had an average activity of
5% per gram of cell pellet and the non-illuminated had about 1% ........... 181

Figure 4.3 Effect of method of lysis on PAM activity measurement -Cells
illuminated by total white light or grown under non-illumination conditions were
either Iysed by sonication (DSL and LSL)or chemically (DCL and LCL).

DSL— sample covered (dark) and lysed by sonication

DCL- sample covered (dark) and lysed chemically

LSL- illuminated by light and lysed by sonication

LCL-illuminated by light and lysed chemically .......................... 182

Figure 4.4 The effect of UV radiation on the activity of pure PAM. PAM (50 pg)
was incubated with 1 mM of a-phenylalanine at room temperature for 1 h and 24
h respectively. UV light at 254 nm (left) illuminated one set of reaction and the
other was kept under normal room light (right). There is a slight increase in
activity under UV light illumination. Two samples were exposed to light (120, 65
W light bulb) and a control covered and incubated overnight. The results show no
significant difference between uncovered (C) and covered (D) samples ...... 183

Figure 4.5 The effect of sonication on the activity of pure PAM. Samples kept in
ice (ICE) showed no activity. Samples sonicated for a total of 5 minutes (purple)
had 1.4% mol conversion. Control samples incubated for 1 hour at 31 °C under
light illumination (yellow) and dark/covered had about 3.2% mol conversion. . 184

Figure 4.6 Shows the effects of sonication on PAM activity. PAM was first
sonicated for 5 min before addition of substrate and incubation for 1 hour. The
control was incubated for the same time and temperature and time without
sonication ...................................................... 1 85

Figure 4.7 Shows the averaged time course assay trend of the variation of PAM
activity under light illumination (square) and non-illumination (circle) ........ 186

Figure 4.8 Monitoring the growth of E.coli cells transformed with the pam gene
under various light conditions. The cultures illuminated with total white light
without any filters had higher ODeoo (.) before the saturation phase. The covered
cultures and those exposed to light in which the red and/or IR radiation had been
cut off showed reduced growth during the log phase .................... 187

Figure 4.10. The conversion of trans-cinnamic acid to cis-cinnamic acid under
ultraviolet light .................................................. 242

Figure 4.11 The equilibrium between a-phenylalanine, trans-cinnamic and I3-
phenylalanine ................................................... 242

xvii

Figure 5.1 The two proposed mechanisms of PALs and HALs. On the left shows
the aromatic ring of the substrate attacking the MIO and on the left the amine
group of the substrate attacks the MIO. In the case of PAL trans-cinnamic acid is
generated as the product .......................................... 220

Figure 5.4 The expected intermediates of the substrate during the course of the
reaction and the expected chemical shifts. The mechanism is based on the
assumption that the amine of the substrate is the nucleophile. Tyrosine 322 is
believed to be the base that de-protonates the B-hydrogens (refer to Chapter 5
for more details). The reaction goes through three intermediates II, III and IV
before the product V is formed ...................................... 283

Figure 5.5 The schematic reaction mechanism proposed if the aromatic ring of
the substrate is the nucleophile. The ipso carbocation (II) is expected to be very
short lived, although its detection will provide valuable information. The phenyl
ring attacks the MIO to form the complex II. The tyrosine residue de-protonates
the B-H to form intermediate Ill. The free amine picks up the proton from the
tyrosine to form IV which is followed by the re-aromatization of the substrate and
the elimination of free NH3, which rebounds to the C3 position to form the
product ........................................................ 283

Figure 5.6 130 NMR of the 13C labeled substrate (A), expanded aliphatic region
(B), expanded aromatic region and expanded carbonyl region (C) and the
carbonyl region (D), showing the splitting pattern, ...................... 288

Figure 5.7 Array of 130 NMR spectra for the PAM reaction with isotopically
enriched L-d-phenylalanine. The lowest spectrum was acquired before adding
the enzyme. One of every 3 scans is shown out of a total of 33 scans ....... 230

Figure 5.8 130 NMR Spectra of the product after 16 h incubation with PAM. (A)
full spectrum, (B) an expanded aromatic region, (C) and expanded carbonyl
region and (D) an expanded aliphatic region .......................... 232

Figure 5.9 Comparison of the 13C NMR spectrum of the biosynthetically made
product (A) versus the authentic B-phenylalanine (B) .................... 233

Figure 5.10 130 NMR of the PAM reaction product. (A) 13C labeled substrate. (B)
Product after overnight incubation with PAM. (C) Product after work-up by
precipitating the enzyme .......................................... 234

Figure 5.11 ”C NMR for cis-cinnamic acid (A), biosynthetically synthesized
product (B) and trans-cinnamic acid (C) .............................. 235

xviii

Figure 5.12 ”N NMR for [u,‘3c9, 15Nj-phenyiaianine with PAM. The enzyme
and substrate were mixed in an NMR tube and NMR spectrum taken (A) before
adding PAM and (B) 15 min after reaction initiated ...................... 237

Figure 5.13 The 15N NMR spectrum of isotopically enriched substrate incubated
with wild type PAM over 16 hours. Initially 3 peaks are distinguishable at 11 ppm,
36 ppm and 44 ppm .............................................. 237

Flgure 5.14 The plot of relative rates of formation of the substrate (blue), 8-
(purple) and trans-cinnamic acid (yellow). The mass balance is referenced to the
substrate (5 mM). At equilibrium the a- and B- phenylalanines have the same
(~20% each) .................................................... 238

Figure 5.15 The role of PAM as an aminating enzyme. The o- and B-amino acids
are formed from their corresponding cinnamates ....................... 241

Figure 5.16 The potential function of PAM as a transaminase based on the
functionality of TAM a mechanistically similar enzyme ................... 242

xix

LIST OF SCHEMES

Scheme 1.11 Synthesis of (2S,3R)-, (2R,3S)-N-acetyI-B-methyl ester
racemate ....................................................... 21

Scheme 2.2 Coupling of baccatin III with a B-lactam produces Taxol in two steps;
(i) NaH, THF, B-lactam and (ii) HF, pyridine. The B-lactam was synthesized in
seven steps ..................................................... 59

Scheme 2.3 Biosynthesis of paclitaxel. The proposed mechanism for the
oxetane ring formation is shown in the inset .......................... 62

Scheme 2.4 Biosynthesis of C-1027 an antibiotic and antitumor agent isolated
from Streptomyces globisporous. The first step of one of the biosynthetic
branches to C-1027 is catalyzed by tyrosine aminomutase which converts a-
tyrosine to B-tyrosine (inset) ......................................... 65

Scheme 2.11 Biosynthesis of taxol from a-phenylalanine which is first converted
to B-phenylalanine by the enzyme phenylalanine (PAM) (boxed).

Scheme 2.14 The synthesis of B-allylglycine for use as standard of the PAM a-
allylglycine reaction ............................................... 96

Scheme 2.15 Synthesis of (E)-5-aminopent-3-enoic acid .................. 99

Scheme 3.1 The reaction of cysteine with the MIO moiety of PAL or HAL at pH
10.5. The reaction is irreversible in the absence of oxygen and is stable under
aerobic conditions ............................................... 118

Scheme 3.3 The reaction of cysteine with the MIO moiety of PAL of HAL at pH
10.5. The reaction is reversible in the absence of oxygen and stable under
aerobic conditions ............................................... 121

Scheme 3.4 The PAM reaction is assayed by incubating the enzyme with the
substrate at 31 9C. The reaction is quenched with 1 N NaOH before adding acetic
anhydride followed by diazomethane after extraction with ethylacetate at

pH 2 ......................................................... 124

Scheme 3.6 Shows the formation 3, 5-dihydro-methylidene-4H—imidazol-4-one
prosthetic group (far right) which occurs in the active site of HAL from P. putida.
The alanine-serine-glycine triad undergoes cyclization and dehydration to form
the MIO motif ................................................... 127

Scheme 3.22 A summary of the epimerization reaction catalyzed by the

phenylalanine racemase, the imine intermediate attacks a proton from both faces
to form a racemic mixture of L and D phenylalanine ..................... 144

XX

Scheme 3.25 The proposed formation of the MIO from the cyclization and
condensation of the ala—cys-gly triad. The reaction results in the loose of one
water molecule and one hydrogen sulfide molecules .................... 149

Scheme 5.2 The 1, 2 shift of the amine group is initiated by the binding of the
amine of the substrate to the catalytic MIO cofactor. The mechanism is based on
the crystal structure of SgTAM ...................................... 221

Scheme 5.3 General reaction of ammonia lyases. The aromatic amino acids are
non-oxidatively de-aminated to their corresponding amino acids ........... 222

xxi

LIST OF ABBREVIATIONS

PAM ................................... phenylalanine aminomutase

PAL ................................... phenylalanine ammonia lyase

TAL .................................... tyrosine ammonia lyase

HAL .................................... histidine ammonia lyase

TAM ................................... tyrosine aminomutase

LAM ................................... lysine aminomutase

MIO .................................... 3,5-dihyro-5-methylidene-4H-
imidazol-4-one

E. coli .................................. Escherichia coli

IPTG .................................. lsopropyl galactopyranoside

SDS ................................... sodium dodecyl sulfate

PAGE .................................. polyacrylamide gel
electrophoresis

ASG ................................... alanine-serine-glycine triad

GC/El-MS ............................... coupled gas chromatography
and electron- impact mass
spectrometry

Elcb ................................... first order elimination reaction
that proceeds via a carbanion
intermediate.

$1760, A, T and G ......................... mutant of PAM where serine-

176 has been replaced by
cysteine, alanine, threonine
and glycine.

Y322F, H ................................ mutant of PAM where
tyrosine-322 has
been replaced by
penylalanine or histidine

xxii

C107A, H ..................... mutant of PAM where cysteine-107 has
been replaced by alanine or histidine

ATP ........................ adenosine triphosphate

EtOAc ...................... ethyl acetate

1H- and 2H-NMR .............. proton- and deuterium-nuclear magnetic
resonance,

kDa ........................ kilodaltons

k.i.e ....................... kinetic isotope effect

LB ......................... Luria-Bertani medium

MWCO ...................... molecular weight cut-off

c1 ........................... alpha

[3 ............................ beta

p ............................ micro

mL .......................... milliliters

pL ........................... microliters

pg ........................... microgram

6 ............................ delta

ppm ......................... parts per million

W ........................... watt

Nm .......................... nanometer

mm .......................... millimeter

cm ........................... centimeter

h ............................ hour

5 ............................ second

PCR ......................... polymerase chain reaction

xxiii

dNTP .......................... deoxynucleotide triphosphate

MQ H20 ........................ milliquire water

Sg ............................ Streptomyces globisporous
T. brevifo/ia ..................... Taxus bre vifo/ia

2’-Ado ......................... 2’-adenosine

SAM ........................... S-adenosine methionine
PLP ........................... pyridoxal pyrophosphate
GTP ........................... guanidine triphosphate

A . G ............................ Gibbs energy change

Vmax ........................... maximum velocity

KM .............................. Michael-Menten constant
Kcat ............................ catalytic turnover

Vrel ............................ relative velocity

Vo . . . ......................... initial velocity

FW ............................ formulae weight

UV ............................. ultraviolet

MHz ............................ megahetz

Ni-NTA ........................ . nickel nitrilotriacetic acid
RIPL ............................ arginine lsoleucine proline lysine
mM ............................ millimolar

p .M ............................ micromolar

xxiv

CHAPTER 1

Assessment of the Cryptic Stereochemistry of Phenylalanine
Aminomutase: Retention of Configuration at the Migration Termlnl.

1.1 INTRODUCTION

Aminomutase enzymes typically catalyze the isomerization of o-amino
acids to B-amino acids; however, there are a few that catalyze the migration of a
terminal amino group position at the 6- and 5- carbon of appropriate amino acids.
Of the seven aminomutases identified so far there are five distinct proposed
mechanisms. The first class include D-lysine-5,6-aminomutase, D-ornithine-5,4-
aminomutase and leucine-2,3-aminomutase which utilize adenosylcobalamin and
pyridoxal phosphate (PLP) as cofactors and involve the cleavage of the C-Co to

generate a radical.“8 Another mechanistic class of aminomutases is represented

55-60

by lysine-2,3--aminomutase9'54 from Clostridium subterminale, which belongs

to a group S-adenosylmethionine (SAM)-dependent enzymes which carry an

iron-sulfur (4Fe-4S) cluster and also require PLP; a 5'-deoxyadenosyl radical is

60-77

generated during the reaction. A third class uses only PLP without

generation of radicals. For example, glutamate-1-semialdehyde aminomutase
catalyzes the conversion of glutamate-1 -semialdehyde to 6-aminolevulinic acid,78

which is the universal precursor for the biosynthesis of heme, chlorophyll, and

79-83 84-88

other pyrroles. A fourth class includes tyrosine-2,3-aminomutase from

Bacillus brevis that only requires ATP as a cofactor. The final mechanistic class

includes tyrosine-2,3-aminomutase from Streptomyces globisporous (SgTAM)
and phenylalanine aminomutase (PAM) from Taxus.
The stereochemical course of the Iysine-2,3-, tyrosine-23, arginine-23,

89,90 Th

and the o-lysine-5,6- aminomutase reactions have been evaluated. e

vicinal transfer of the amino group of each substrate was shown to invert the
configuration at the receiving carbon, replacing one of the diastereotopic
hydrogens formerly at this position. Of these isomerases, the stereoselectivity of
the reciprocal hydrogen transfer is known for only the arginine-2,3- and lysine-
2,3- aminomutase reactions, wherein the configuration at the receiving prochiral
carbon is inverted. The stereochemistry of the arginine-2,3-isomerase reaction
was assessed in vivo in Streptomyces griseochromogenes by feeding
stereoisomers [2,3,3—2H3]-,[3,3-2H2]-, and [2-2H]-arginines.91 The results
revealed the complete retention of the original o-hydrogens with migration of one
to the o-position, as well as partial loss of the original o-hydrogen presumably
due to arginine racemase activity. (3F?)- [3-2H1-and (38)-[3-2H]-arginines were
synthesized unambiguously and used to determine that the pro-3R hydrogen of
a-arginine migrates to the o-position (C-2).

The cofactor requirements of arginine-2,3-aminomutase (AAM) from
Streptomyces have been empirically determined to be (S)-adenosylmethionine
and pyridoxal phosphate. However, the catalytic mechanism likely involves

homolysis and free-radical coupling processes that initiate the removal of

relatively non-acidic alkyl hydrogen (pKa 2 45) at CB from the arginine substrate.

Arginine is structurally similar (from CC. to C5) to the a-ornithine and a-lysine

2

substrates of their respective aminomutases, whose mechanisms are radical
mediated. Furthermore, o-arginine lacks electronic stabilization, which could be
achieved by the presence of, for example, an adjacent aromatic or allylic group

that would increase the acidity of the hydrogen at CB in a heterolytic reaction. A

1 1
5N, 3

series of C-Iabeled d-arginines were fed to the Streptomyces in order to

examine the mechanism of L-B-arginine formation in the biosynthesis of the
antibiotic blasticidin S. Analysis of the derived antibiotic by 1SC NMR

spectroscopy revealed intramolecular migration of the amino group from the o- to
the B-carbon, revealing the presence of an arginine-2,3-aminomutase.
The multifunctional phenylalanine aminomutase (PAM) from Taxus

catalyzes the vicinal exchange of the amino group and the pro-(38) hydrogen of

92-101

(2.5')-o-phenylalanine to produce (3R)-B-phenylalanine. While the migration

of the amino group from 02 to C3 of the product was already known to proceed

98-1 00

intramolecularly with retention of configuration, the stereochemistry of the

hydrogen transfer remained unknown. The stereochemistry of the amine group at
the ca position was established as n by incubating PAM with (emu-”ci-

phenylalanine, then converting the product to the 1-(S)-camphanate methyl ester,
and finally comparing its retention time to that of authentic N-[(1'-S)-campanoyl]-
(3R)-B-phenylalanine methyl ester on radio-HPLC. The R configuration of the [3-
phenylalanine catalyzed by the aminomutase on the paclitaxel synthetic pathway
is consistent with the configuration of the amino group found in paclitaxel. This

supports the premise that B-phenylalanine is indeed an intermediate on the

paclitaxel pathway. Use of (S)-[2-15N, ring-ZHsj-phenylalanine demonstrated that
the amine group is transferred intramolecularly during the course of PAM
catalysis. When (2S,3S)-[ring, 2,3-2H7]-phenylalanine was used as a substrate,

no deuterium was detected at the C3 position, but a 2,2-dideuterio B-product was
confirmed by GC/MS analysis suggesting that only the (3S)-deuterium migrated
from the C3 to the C2 position (Figure 1.1 B).

Not only is the retention of stereochemistry at the migration termini of the
amino group an uncommon characteristic of all the aminomutase mechanisms

described, but the stereochemistry of the product is opposite that of the (38)-
product made initially in the mechanistically-related TAM reaction.86 However,

despite this apparent divergence in stereochemistry between the aryl amino acid
aminomutases, both the lack of cofactor dependency and similarity of the amino
acid sequences between PAM and TAM (from Sfreptomyces) (56% amino acid
similarity) reveal that these enzymes belong to the same family, which includes
several ammonia lyases (Figure 1.3). The lyases share a characteristic active
site motif, ASG (also present in the aminomutases) that autocatalytically

rearranges to form a functional 3,5-dihydro-5-methylidene-4-H-imidazol-4-

one(MlO) prosthesis. The MIO facilitates the elimination of NH3 group and H+

from the arylalanine substrate to form an aryl acrylate product in the lyase
reaction. The MIO moiety purportedly couples with the substrate by electrophilic
attack on the ortho-carbon of the aromatic ring or by direct electrophilic capture

of the amino group (Figure 1.2). Similar MIO involvement is proposed in the

. . . . . . +
aminomutase reaction mechanism where, instead, the vucrnal NH3 and H

interchange intramolecularly and rebound to the phenylpropanoid to complete the

reaction cycle forming the B-amino acid product (Figure 1.1A).

O
COO pAM ‘ COO
—_>

  

H H'
@NH3
(S)-o-phenylalanine (FOB-phenylalanine
O
H D O H ‘\NH3COG
B I \ Wgoo pAM I \ O
// H3gD // 00") D
05 D5
69
D H O D NH3 6

 

C W330 PAM E:)§<coo
H H
H3%

Flgure 1.1 The reaction of PAM with a-phenylalanine showing the retention of configuration of
the NH2 group migrating from C2 to CS (A), confirming that only the pro-(3R)-hydrogen migrates
to the CZ position (B and C).

 

amino grouFEctivation
by MIO

 

n1
o-complex with MIO

Figure 1.2 The proposed mechanism of PAM involving the aromatic ring as nucleophile (left) or
the amine as nucleophile (right).

   
   

       
   

    
  
 
 

 
     
 
    
 

PAM_T.cana :
PAM_T.chin :
PAL_G.bilo :
PAL_P.sylv :
PAL_A.thal :
PAL_R.toru :
TAM_S.glob :
HAL_P.puti :
TAL_R.spha :
* 260

PAM_T.Cana : TKPSVVARIGDDVE--- P"~
PAM_T.Chin : lIGKPSVIARIGDDVE--- PA'E-L
PAL_G.bilO : 1L GRPNSKVRTRDGTE---HSG EflL
PAL_P.sylV : GRPNSRVRSRDGIE--- SG-EFL
PAL_A.thal : RPNSKATGPDGES---
PAL_R.toru : 'DSKVHVVHEGKEKI Y
TAM_S.glOb : EG—--YVLRDGRP--- ET
HAL_P.puti : IfSL L GEG--—KARYKGQw-—- SA
TAL_R.Spha : OGRGD--FLDRDGTR--- DG

6 2

* 280 * 300 * 320 *

PAM_T.Cana : SRVG P--F1LO'KEGLALVNGTSFAT STVMYDiEVLLLLVE GlF E l URE- V
PAM_T.Chin : SRV RP—-F1L0'HEGLALVNGTSFAT STVMYD‘ VLLLLVET G FOE I GRE- .
PAL_G.bilO : KQVG K-PFoLo'KEGLAIVNGTSVG SIVCFDQEVLAVLSE NS F E V I‘iKP-Iliit‘
PAL_P.SylV : KKVG EK-PFJL EEGLAIVNGTSVG SIVCFD VLALLSE S IF E * GKP- ‘
PAL_A.thal : EKAG STGFFILO'HEGLALVNGT VGSGI SMVLFEANVQAVLAE S F . FKP— .
PAL_R.toru : ALFN EP-- 'KEGLSLVNGT VSAS' TLAL LSLLSQS i1" 12- GI'l-GSFI -
TAM_S.glOb : AERG P--L9LR HEGLALINGTSGMTG GSLVVG LEQAQQAEI LIE-V'GSTSP.
HAL_P.puti : AVAG P--L L--KEGLALLNGTOASTAEELRGLFY.EDLYAAAIACGG S ‘ GSRS'F -'
TAL_R.spha : RRG MIL-IP-LAL‘i-TIGT LVNAHACRHLGNWAVAET L ..

 

.- — ‘30-...—

Flgure 1.3 A partial sequence alignment of PAM__ T. canadensis with other MIO carrying enzymes
PAM_T. chinensis, PAL_G. biloba, PAL_sy/vesfris, PAL_A. fhaliana, PAL_Ff. toruloides, TAM_S.
globisporous, HAL_P. putida and TAL_FI. sphaeroides.

On the surface, the net vicinal rearrangement reactions of the 2,3-
aminomutases appear similar. However, there is clear mechanistic diversity
among the B-isomerases. Therefore, unveiling the complete stereochemistry of
the reaction mechanisms will help to identify the determinants that sort the
aminomutases into coherent sub classes. In this study, the cryptic
stereochemistry of the PAM reaction involving the hydrogen transfer from C3 to
CZ of the phenylpropanoid substrate is defined. A deuterium label tracer method
is described for the measurement of the overall kinetic isotope effect of the CB-H
bond cleavage. The exchange of the transient hydrogen with protons from an
alternative source was also investigated in deuterium-labeling studies, and the
equilibrium constant of the PAM reaction was measured to assess the difference

in free energy between the reactant and the product.

1.2 RESULTS AND DISCUSSION
1.2.1 Hydrogen Exchange at 02 of the PAM Reaction Product.

The hydrogen exchange in the PAM reaction was tracked as a function of

time to further evaluate cryptic aspects of the mechanism. [ring, 2,3,3-2ng-(ZS)-
a-Phenylalanine, at saturation (500 pM), was incubated with PAM (KM z 45 pM)

in buffer dissolved in H20 over a period of 50 h. After 0.25 h, 0.2 mol% (0.5 nmol)
of the substrate had converted to B-phenylalanine that was comprised of ~57%
D7-isotopomer and ~43% of Dg-isotopomer (Figure 1.3) as determined by GC/El-
MS analysis. The D7 species contained the intact phenylpropanoid fragment ion

(m/z 185), and a fragment ion derived from C2-C3 bond fissure m/z 154 (a D6
species, no 05 species were identified) indicated that the deuterium resided at

the phenyl (D5) and benzylic (D1) carbons (Table 1.1B). Thus, the H- for D-
replacement was confirmed to have occurred at CZ, and not at CS, nor in the
aromatic ring. During the period from 0.25 to ~Z h, the isotope exchange rate
constant was at 0.52 h'1 while the product abundance increased from 0.2 mol%
to 0.8 mol% relative to substrate (Figure 1.3). The H/D exchange rate decreased
and finally reached equilibrium (~75 % H- for D- replacement) at longer
incubation times, while the product increased from 0.8 mol% to 10 mol% relative
to substrate between 2 and 50 h.

The origin of the proton source in the exchange experiment was
ascertained through a reciprocal experiment conducted by incubating PAM with

unlabeled o-phenylalanine in buffer, wherein the H20 was replaced by 020. After

a 12 h incubation, the derivatized B-phenylalanine product isolated from this
enzyme-catalyzed reaction was comprised of ~70% D1- and ~30% Do-

isotopomers and confirmed the extent of hydrogen replacement observed in the
previous experiment with unlabeled solvent (Table 1.1C). Evaluation of the 8-

amino acid isotopomers by GC/El-MS fragmentation confirmed that the
deuterium in the D1-species was at C2 (Table 1.1C). Furthermore, if the

deuterium exchange described above resulted from the in situ chemical N-
acetylating and methyl esterification steps used to derivatize the amino acids,
then complete replacement of deuterium by hydrogen would have been
anticipated, particularly for samples in which the B-amino acid product was least

abundant; yet, this was not observed.

 

9°
0"
.5
N

9°
C
lanine

i
A
o

N
0'!
i
(D

T
O)

 

A
mol% B-Phenylalanine Relative a-Phenyla

 

\
\
i

N

 

 

Ratio of D7 to 08 lsotopomers of Biosynthetic B-Phenylalanine
N
o

 

.0
o
o

o 10 20 30 4o 50
Time (h)

Figure 1.4 Hydrogen exchange at the C2 position

Table 1.1 A summary of the H/D exchange during the PAM reaction. Diagnostic ions resulting
from electron impact-mass spectrometry fragmentation. Shown are mass spectral fragments after
bond fissure at sites 'a' and 'b', yielding diagnostic ions of the corresponding N-acetyl-o-
phenylalanine methyl ester.

3
AS’NH x

@{yé3oow3
V \

HN + NH

x l
' COOCH3
x
Yz

Z-(S)-a-phenylalanine Ion (m/z) observed by El-MS analysis

 

 

Hi

9
coo
m/z178 (Do), x,z,v = H m/2148 (Do)
NH3 X: H
e
D 90 e
\ ‘ . coo
|// ”3% "’o m/2185 (07) x, z = D, Y = H, ring = D5 m'/2154 (De)

5 m/z186 (Do), X, Y, Z = D ring = 05

 

U

 

H

9
COO
m/z178(D0)X,Y,Z=H m/z148(D0)
gm; m/z179(D1)X,Y=H,Z=D X=H
0‘00 6
" coo
m/z179(D1):X,Y,Z.-.H m/z149(D1)
8H3 m/z180(DZ):X,Z=D,Y=H X=D

 

 

 

 

 

 

10

1.2.2 Complete Stereochemistry of the PAM Reaction.
The 1H-NMR chemical shift values of the diagnostic protons at CZ of [3-
phenylalanine were measured and observed at 6 = 5.43 (doublet of doublets), for

CBHNH, and at 5 = 2.92 (doublet of doublets) and 2.82 (doublets of doublet) for
CaHH (Figure 1.5B). The diagnostic protons at CaHz in a mixture of unlabeled

authentic N-acetyl (3R)-B- and (ZS)-d-phenylalanine methyl esters (each at ~200
pM) dissolved in [2H3]-ethyl acetate, and were found to be well-resolved (by A6
0.24) from the chemical shifts signals of the protons at C3 (i.e., the diastereotopic
benzylic protons (CBHz) of o-phenylalanine at 5 = 3.08 (doublet of doublets) and

3.15 (doublet of doublets) (Figure 1.50). The resolution between the d- and B-
phenylalanine proton chemical shift signals indicated that the signals for both

compounds could be measured simultaneously. Therefore, the need to
chromatographically-separate the [2H]-labeled a- (substrate) and B-(biosynthetic
product) amino acids in the biosynthetic assay prior to assessing the

stereochemistry of the CZ deuterium in the [2H]-labeled biosynthetic product by

zH-NMR was precluded. The relative configuration of the prochiral hydrogens at
C2 of B-phenylalanine were assigned by employing the racemate of N-acetyl
(ZS,3R)- and (ZR,3S)-[2,3-2H21-d-phenylalanine methyl ester that was
synthesized by stereo specific reduction (palladium/D2 gas) of an N-acetyl trans-
enamine methyl ester (Figure 1.12); the (ZS)- and (ZR)-deuteriums in the
enantiomeric pair are spectroscopically equivalent. 2H-NMR analysis of the

synthetic dideuterio product, dissolved in ethyl acetate, revealed chemical shift

11

resonances at 6 = 5.42 (CBDNH, singlet) and at 2.82 (CODH, singlet); the latter

signal corresponds to the deuterium at the (ZS)- and (2R)-positions of the

enantiomeric mixture (Figure 1.6).

12

A

_ll.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I AL All .iL -
I I I 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
7.0 6.0 5.0 4.0 3.0 2.0 1.0
"Ifi"'lF"'l""l""l"
3.10 3.00 2.90
B
- L L “.2 ....L
I fir T I I I I I I I I 1 I I I I I I T I I I I I I I I I I I I I I I I I I I I
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
I I I I I I I I I I I I
3.00 2.90 2.80
C
i A t I . Au M J. _
' ' ' ' I ' ' ' ' I ‘ ' I I ' ' ' ' I ' ' ' ' I ' ' I ' ' I
7.0 6.0 5.0 4.0 3 0 2.0 1.0

 

 

 

 

3.20 3.10 3.00

 

IIIIIITIIIIIIIIIrIIIIlIII

2.90

2.80

 

 

Figure 1.5 1H NMR for d-phenylalanine (A), B-phenylalanine (B) and d- and B-phenylalanine, the
amino acids were derivatized to their mixture of N-acetylated methyl esters. The expanded
regions showing the splitting patterns are shown in the inserts below the full spectra.

13

 

 

 

 

 

 

A JL A
F l
5.0 0.0
B l M l l
' ' ' ' T ' ' ' ' l ' ‘ . ' I ' ' ' ' F
4.0 3.0 2.0

Figure 1.6 A. 2H-NMR for an enantiomeric mixture of [2.3-2H2]-(ZS,3R)- and (2R,3S)-I3-
phenylalanine as N-acetylated methyl esters. The peaks are observed at 6 = 2.82 and 5.32 ppm
for d- and B- 2D respectively. B. The 2H-NMR of [3,3-2H21-d-phenylalanine derivatized as the N-
acetylated methyl ester.

1
-> COOCH3 -> OOCH3
D NHAC AcHN D
D H H D
-> Ph "> Ph

Figure 1.7 The Newman projects of diastereoisomers (28,3R)-, (2R, 3S)-[2-,3-2H2]-N-acetyl-
phenylalanine methyl ester.

14

O

D \D
HN O

y;

Biosynthetic product

 

 

 

 

O
/U\N|;|D O
3 ,CH3
., 0
(iii) D 9H
(iv) ‘/ \
O
.H O /U\
. ,CH3 Nil-jH O
-, O ' CH
HN H 0’ 3
F" H ’H

 

 

 

‘.—L _
L l 1 l I l\l i l l j

350 3.00

Figure 1.8 Study of the complete stereochemistry at the CZ position of the B-product with
reference to the migratory hydrogen from the C3 position. (l) [3,3- H2]-phenylalanine as

substrate, (ii) the biosynthetic product, (iii) the aunthentic [2.3-2H21-phenylalanine and mixture of
natural d- and B-phenylalanine. All the amino acids were derivatized to their methyl esters.

A 2H-NMR spectrum of the [3, 3-2H2]-labeled substrate was acquired for

referencing the chemical shift signals of the unused substrate in the enzyme
assay to examine the stereochemistry (Figure 1.6B). The labeled o-amino acid

was incubated for 10 h In assay buffer without PAM, and derivatized and isolated

15

 

like the amino acids in the catalytic assays. The derivatized amino acid showed

deuterium chemical shift resonances at 6 3.09 and 3.15 for the diastereotopic
deuteriums (Figure 1.8.l) in the 2H-NMR spectrum; the chemical shifts were

identical to those of the unlabeled isotopomer that was analyzed by 1H NMR
(Figure 1.8.iv). Incidentally, D-D spin couplings are about 40 times smaller than
H-H spin couplings; therefore, the resolution of peak splitting caused by this
coupling is not observed in 2H-NMR, and the deuterium signals appear as
singlets.

[3.3-2ng-(2S)-d-phenylalanine (5mM) was incubated for 10 h with 50-fold
more PAM enzyme than used in general assays. The reaction progress of the

enzyme assay was monitored by GC/MS-El for this investigation, and the

reaction was stopped when it was estimated that sufficient product had been
made and was comprised of enough [2,3-2Hj-B-phenylalanine isotopomer for 2H-
NMR detection. These conditions were met just as equilibrium was reached, i.e.,

the abundance of substrate and product were near 50:50. Had this reaction

continue any longer at equilibrium, all of the migrating deuterio group will have
been washed out (Figure 1.9). 2H-NMR analysis of the [2H]-labeled p-
phenylalanine isotopomer derivatized and isolated after incubation revealed
chemical shifts at 6 = 5.43 (HNCBD) and at 6 = 2.82 (HCaD) (Figure 1.8.iii).

These signals were identical to those of the authentic deuterated racemic B-

phenylalanine synthesized earlier (Figure 1.6A). This NMR data coupled with the

known, exclusive (3R)-stereochemistry of the biosynthetically-derived [2H]-B-

16

phenylalanine product, establishes the biosynthetic product as the (28, 3R)-

enantiomer.

Further analysis of the 2H-NMR spectrum of the derivatized biosynthetic
product (Figure 1.8.iii) showed that when the integral of the peak area for the
resonance signal at 6 = 5.43 (CBD) was set to the expected value of 1.0

deuterium, the relative abundance of deuterium at C2 was a fraction (~0.30) of its
expected value of 1.0 (Figure 1.9). This fractional loss of deuterium supports the
level of hydrogen exchange observed in the isotope replacement assays

conducted earlier. This isotopic ratio was confirmed by GC/El-MS analysis (Table

1.10) of the product; the relative diagnostic ion abundances were at 100% (D1
species) and 28% (02 species) (Table 1.1D), after correcting for the mass

contributions of natural abundance 13C.
A peak area of 1.8 was assigned to the chemical shift signals corresponding
to the two diastereotopic deuteriums at C3 of the remaining [3.3-2H2]-Iabeled

substrate in the enzyme assay, above. Comparing the integral of 1.8 to the

integral of the signal (set at 1.0), in the same spectrum, that corresponds to the
peak area of the signal corresponding to the one deuterium at C3 (CBDN) of the

biosynthetic B-amino acid, indicates an approximate substrate to product ratio of
48:52 (Figure 1.10). lmportantly, if the D- for H- exchange observed in

biosynthetic product, resulted from the chemical derivatization of the amino

acids, then racemization at Co. of B-phenylalanine would have been highly

l7

anticipated. This effect, however was not observed in the 2H-NMR spectrum of

the biosynthesized [2H]-B-phenylalanine derivative (Figure 1.8.ii).

D at C3 EtOAc

o tCZ
[2,3-2H21-(ZS,3R)-B-Phe a

[2,3-2H2]-(ZS,3R)-B-Phe

 

 

l I I I I I I f I I I I I I r I I T l I I I r I l I 1 l I
I F I I I l I

6.50 6.00 5.50 5.00 4.50 4.00 3.50

Flgure 1.9 Assessment of the equilibrium conditions of the PAM with respect to the unreacted
substrate and product. Dideteurated d-phenylalanine was incubated with PAM at 30 °C until the
reaction reached equilibrium (17 h).

1.2.3 The Rate Determining Step on the Isomerization Pathway.

An equal mixture of unlabeled- and perdeuterio-(ZS)-d-phenylalanines (at
saturation) was incubated for 3 h with PAM under typical conditions, and the
reaction was processed for analysis as described previously. The resulting
biosynthetically-derived B-phenylalanine isotopomers (~0.7 mol% relative to

substrate) were comprised of 66 mol% of Do product and 34 mol% of a mixture of
D7 and D8 product. This suggests a primary isotope effect on Vmax/KM of

approximately 2.0 :I: 0.2 for the removal of the pro-3S hydrogen, which is likely a

rate-determining step of the catalytic cycle. Notably, no D1-Iabeled [3-

18

phenylalanine was detected, indicating that intermolecular deuterium crossover

from the [2H8]-d-phenylalanine to the Do B-isomer was below detection limits.

1.2.5 Equilibrium constant measurements

The equilibrium of the PAM reaction was measured by incubating PAM
(100 pg) with (ii-phenylalanine (100 pM) in 10 mL of 50 mM sodium phosphate
buffer pH 8.5 at 31 °C. Samples (1mL) were collected at time intervals ranging
from 15 min to 24 h. The mol% of substrate and the B-phenylalanine product
derivatized as their N-acetyl methyl esters were monitored by GC/MS analysis

(Figure 1.9). An equilibrium (Keq) of 1.1 favoring the B-isomer was assessed.

 

 

 

120
a)
.E 100 t;
C 0
§ 80
m ’ e
5 60
gr} 9 ‘2 9
o 40
o\° o 0
6
E 20 00

o If

 

0 500 1 000 1500 2000 2500 3000 3500

Time (min)

Figure 1.10 Measurement of the equilibrium constant of the PAM reaction with the natural
substrate. PAM (100 pg) was incubated with 100 pM substrate and samples were at set time
points and assayed as above. The d-substrate change over time is shown in black square and
the B-product in open circles.

19

1.3 DISCUSSION
1.3.1 Hydrogen Washout during Isomerization Chemistry.

The mechanism of the phenylalanine aminomutase reaction is considered
to proceed, in part, similar to that proposed for the reaction of the related
ammonia lyases, as mentioned previously. In terms of reaction chemistry and
substrate specificity, the phenylalanine ammonia lyase (PAL) provides a practical
model upon which to base the PAM reaction. The ASG amino acid sequence
motif of PAL is proposed to form an MIO moiety in the active site that provides a
similar function as a Lewis acid to promote the elimination of ammonia from the

phenylpropanoid (Figure 1.11). This elimination process begins by removal of the

pro-(3S) hydrogen by putatively a 137His residue of PAL from Rhodosporidium

102

toru/oides (Figure 1.11.iv), followed by E1cb-type displacement of ammonia

103,104 (

from the substrate to form trans-cinnamate Figure 1.11.vii). The mode of

MIO-substrate coupling via the aryl ring in the PAL reaction (and other aryl amino
acid lyase reactions) has been widely studied and is strongly supported
empirically, and, therefore, is incorporated in the proposed mechanism for the
aminomutase reaction. Thus, by analogy, the ASG sequence of the
phenylalanine aminomutase from Taxus (50% sequence similarity to PAL from R.
toruloides, ( Figure 1.2) is proposed to also form a functional MIO that assists the

isomerization reaction.100 However, removal of the pro-3S hydrogen in PAM is

107

likely initiated by the conserved Cys residue (see Figure 1.2) that aligns with

20

363Tyr of PAL. Moreover amino acid analysis reveals that no histidine of the PAM

137

sequence aligns with His of PAL from R. toruloides (see Figure 1.2).

H
\ cooe
trans-cinnamate
H

 

 

 

 

\Tyr322
9/
HT” N“\( N \ Acys N \
O Q N Enz HO \ N S OflN O\TW322
)HP' 0C H @H' H ““3 Hi
i WV/ 1 NH3 ii \1/ .
cooe Enz_ GSACYS c009 pAM cooe PAM cooe

R-B—

PAM phenylalanine

syn-periplanar

 

Figure 1.11 The mechanism of PAL R. toluroides (steps vi and v)and the proposed mechanism of
phenylalanine aminomutase (PAM) (steps i-iii). Step (i) is the proposed nucleophilic attack on the
aromatic ring on the methylidene of the MIO, followed by (if) the collapse of the sigma complex

and elimination of NH3 , and step (iii) the rebound of the NH3 at the C3 position. Step (iv) is the
sigma complex formation and (v) is the NH3 elimination as proposed for the PAL mechanism.

Deuterium-labeling studies incorporating GC/MS analysis confirmed that

the pro-(3S) hydrogen of phenylalanine is partially intermolecularly exchanged

with protons from water (pH 8.0) as it migrates to CCl of the product during the

21

isomerization reaction. There are likely two major pathways responsible for the
observed proton dynamics that include H-exchange between 1) substrate and
enzyme and 2) between enzyme and solvent.105 Thus, the extent of deuterium

exchange in the B-amino acid product formed by the PAM reaction from [2H3]-(1-

phenylalanine was evaluated. The substrate remained at saturation (up to 50 h,
<10 mol% product made) to prevent dynamic equilibrium during the time course

of the experiment, and thus minimized contributions from the reverse reaction

(Keq = 1.1). No loss of deuterium was detected in the substrate after 50 h by

GC/El-MS analysis (data not shown), thus confirming that the commitment to the

forward reaction was maintained during the course of the investigation.
Therefore, the 2H-labeled substrate likely transfers the pro-(3S) deuteron

to a proposed general base (tyrosine residue) in the PAM active site. This now-
labeled catalytic residue is either directly accessible by bulk water, and the D- for
-H exchange takes place between tyrosine and the hydrogens of the solvent, or

the tyrosine residue transfers the deuteron to proximate H-exchangeable amino
acids or backbone amides that are reachable by solvent water105. Either of these

transfer events, likely among others, are necessary to account for the H- for D-
substitution that occurs prior to D-rebound since the level of exchange occurs to
a large extent (60-75%) prior to product formation. Hence, the D-rebound to form
substrate must be slower than the rate at which equilibrium is established
between the deuterons transferred from the substrate to PAM than the H- for D-

exchange between the enzyme and bulk water.

22

These aforementioned considerations provide one scenario wherein the
ionizable hydroxyl hydrogen of tyrosine could exchange directly with the protons
of bulk solvent, e.g., solvent has direct access to the active site tyrosine. Thus,

the extent of H- for D- replacement would be expected to be time-independent.
That is, once a deuterium from [2H]-Iabeled substrate transfers to the active site

tyrosine, the H- for D- exchange with solvent would ultimately equilibrate faster
than the slower D/H-rebound. Consequently, an H : D ratio reflective of the two
processes (exchange and rebound) would be established at the start of the
reaction and the ratio of "unlabeled" to "labeled" product2 would partition roughly
according to this ratio; this ratio would be time-independent for the duration of the
reacfion.

However, a transient time-dependent deuterium exchange was observed

early in the PAM reaction (Figure 1.4). Time-dependent hydrogen exchange in

106.107

proteins is common and has been described for various proteins, and the

H/D exchange dynamics have been investigated using 2H-labeling and
electrospray ionization mass spectrometry to probe the rate at which backbone
amide protons undergo chemical exchange with the protons of water.108 Although
amide N-H bonds are covalent and are often well-protected by their hydrogen-
bonding interactions, they, however, engage in continuous exchange with the

hydrogens of water.106

In the deuterium exchange assay with PAM and [2ng-labeled substrate,

D-rebound occurs at ~40% at 15 min, as evidenced by the 3:2 (mol/mol) ratio of

"Unlabeled" to "labeled" B-phenylalanine products at 0.2 mol% abundance

23

 

relative to substrate (Figure 1.4). After several hours, the D-rebound was
comparatively lower at ~25%, and the extent of H- for D- exchange increased
and stabilized at 75%, indicating a 3:1 (mol/mol) mixture of "unlabeled" to
"labeled" B-phenylalanine products at ~10 mol% abundance relative to substrate
(Figure 1.3). This observation is consistent with an initial deuterium "burst," after

the first few round of catalysis that deposits deuterons from the substrate to
active site locations upon isomerization of [2Hg]-d-phenylalanine. The deuteron

transfer from the substrate to the putative active site tyrosine was assessed to be

the slow step in the isomerization reaction, and was based on the primary kinetic

isotope effect on Vmax/Km of 2.0, which was calculated in a competitive assay

with 2H- and 1H-labeled substrates. However, once a deuteron is removed from

the substrate, plausible transfer of this deuteron to other proximate amino acid

105

residues and backbone amides in the active site is likely . Displacement of H-

for D- at these sites on the enzyme would perturb the proton equilibrium between
enzyme and solvent. Conceivably, very early in the reaction, the deuteron that is
shuttled away from the catalytic tyrosine can reasonably be shuttled back part of
the time and rebound to the product, before ultimate dilution with protons from
the solvent. Thus, early in the PAM reaction, relatively less deuterium is lost in
the PAM reaction compared to much later (Figure 1.9). After 2 h, the initial
deuteron “burst" probably approaches dynamic equilibrium with bulk solvent
protons and the rate of deuteron-load onto PAM by the substrate matches the
rate of HID-exchange on the enzyme. The observed 3:1, HID-product ratio

Observed at later times (Figure 1.9) is likely a best approximation for the relative

24

 

rates between the H- for D- exchange and D-rebound. The abundance of B-
phenylalanine increased steadily during this exchange process, indicating that
the hydrogen exchange rate stabilized as a consequence of dynamic
equilibration with active PAM enzyme, and not as a result of compromised
enzyme activity. This observed exchange of substrate protons with bulk solvent
protons contrasts the process in the radical-mediated lysine 2.3-aminomutase
reaction where proton exchange between solvent and substrate is not observed.2

Penetration of bulk phase H2O into the tertiary structure of proteins

109

forming the active site has been demonstrated, whereupon H- for -D exchange

occurs. Therefore, the H- for -D exchange observed between [2H]-labeled

substrate and H20 in the PAM reaction (Table 1.1) suggest that the access of

protons of solvent water to the PAM active site is transiently time-dependent, and
that the exchange rate is fast enough to see significant washout of label from the

substrate.

1.3.2. Stereochemical Fate of the PAM Reaction.

The PAM enzyme catalyzes the conversion of [3,3-2H2j-o-phenylalanine
substrate to a single (3R)-B-phenylalanine isomer as established by a Chiral 00
method,93 (data not shown), 2H-NMR analysis of the [2H]-labeled biosynthetic

product and comparison to authentic stereospecifically deuterium-labeled B-
phenylalanine analyzed by identical methods revealed the product had (ZS,3R)-

stereochemistry. This stereochemical outcome indicated that during the course of

25

the catalytic reaction, the amino group formerly at the (ZS)-position was replaced
by the pro-(3S) deuterium, which migrated from 03, consequently proceeding
with retention of configuration at C2. Reciprocally the amino group migration also
proceeds with retention of configuration at the C3 terminus. The retention of
configuration at both C2 and C3 during the isomerization is a unique mode of
interchange among all of the aminomutases studied (of Table 1.2). To conform
to the stereochemistry observed in the PAM-catalyzed product, the substrate
may bind in the PAM active site with the carboxylate and phenyl ring of a putative

intermediate in a syn-periplanar orientation (Figure 1.10). This orientation
positions the migrating NH3 and H groups of the a-phenylalanine on the same

side of the molecule within the active site for facile exchange that results in
retention of configuration at both receiving carbons (Figure 1.10). The "cisoid"
orientation proposed here is distinctive relative to the anti-periplanar docking

orientation of the d-phenylalanine substrate in the mechanistically-related

110

phenylalanine ammonia lyases (Figure 1.10) and, accordingly, this

conformational difference in binding may likely contribute to the selectivity of the
mutase and lyase reactions.

Notably, although PAM (from a plant) and the tyrosine aminomutase (from
bacteria) originate from disparate sources, these described aminomutases have
56% amino acid similarity (Figure 1.2), and the presence of a signature active
site motif (ASG), present in both, indicates that an MIO prosthetic group is
formed and assists catalysis like in the reactions of the homologous ammonia

lyases. However, these arylalanines aminomutase mechanisms have distinct

26

 

stereochemical differences at the carbon accepting the amino group. The
phenylalanine aminomutase produces exclusively (3R)-B-phenylalanine, even
after several days of catalysis,85 while in contrast, the tyrosine aminomutase
initially catalyzes the conversion of (ZS)-o-tyrosine to (3S)-I3-tyrosine, then
produces the product racemate over time. Therefore, the phenylalanine
aminomutase likely binds its substrate differently than the tyrosine aminomutase,
and/or possibly the catalytic residues of PAM are positioned with an asymmetric
bias that precludes racemization.

Moreover, the hydrogen/deuterium exchange dynamics between
deuterium labeled substrate and product, described in this investigation,

apparently do not epimerize the stereochemistry at C2 during the course of the
reaction. The 2H-NMR spectrum of the [2H]-labeled product showed a deuterium
signal at 6 = 2.82, exclusively, which corresponded to a signal in the 1H-NMR

spectrum for one of prochiral hydrogens at C2 of derivatized authentic unlabeled

(3R)-B-phenylalanine (cf. Figure 1.6), the other signal in this spectrum was
distinctly at 6 = 2.92. No coincident signal at 6 =, 2.92 was observed in the 2H-

NMR spectrum of the labeled biosynthetic product (cf. Figure 1.8.iii).

27

Table 1.2 Summary of the source, substrates, products, cofactor requirement and the
stereochemistry of the products of various aminomutases.

 

Stereochemistry at

 

 

 

 

 

 

 

 

(ZS)-a-leucine

 

 

(3S)-a-Ieucine

 

Amlsnomutase Substrate! Cofactore Product carbon accepting
ource
NH3(H)
*
e H# s” o *H NH3 H#
Lysine-2 3 ”NM-“NW H3N
Clostridium (30% Inverted (inverted)
(ZS)-d-Iysine/Ado Met,PLP (3S)- [5- lysine
O
NH2 NH2 *H NH3 H#
- . H%;Og “NHSH HW;
agggfifc’gsI-1 Inverted (inverted)
(ZS)—d-arginine (3S)-B-arginine
Ht
0 a
Tyrosine-2 3-
. ’ Inverted (unknown)
Bacrl/us HO gHs [4001/9
(25)-d-tyrosine/ATP (36)- B- -tyrosine
Tyrosine-2,3-
Streptomyces N o cofactors Unknown (unknown)
0
H2N 5 3 coo
L sine-5 6- . COO
Ciosmgm H $113 H NH3® NH3 Inverted (unknown)
(3579-05106 (3S, 5S)-diaminohexanoate
Ornithine—Z,3-
Clostridium H2N/5\4/\_/COO SWCOO
NH3 NH2 NH3 Unknown (unknown)
(2R)-d-omithine/AdoCbI, PLP QR 4S)- d-iaminO-pentanoate
G
WOOD NH3
Aggfggiai’his gH3 M9 Unknown (unknown)

 

28

 

1.3.3 Substrate/Product Distribution at Equilibrium.

A general property considered of all amino acids is the
greater strength of the Cp-N bond over the Ca-N bond, as observed for the lysine-
2, 3-aminomutase reaction. The free energy (AG°z —1.4 kcaI-mol") was

calculated to be considerably less than zero, and the forward reaction was
assessed to be largely enthalpy-driven.22 However, in the PAM isomerization

reaction, the product to substrate ratio of nearly 1.0 (Figure 1.9) suggests that the
equilibrium is not principally governed by the difference in ON bond strength

between the d- and B-isomers; apparently, the presence of the aromatic ring of B-

phenylalanine minimizes the difference in CB-N and Ca-N bond energy.

29

1.3.3 CONCLUSIONS

The results of the isotopic labeling studies begin to define the boundaries
of an acceptable mechanism for the PAM reaction processes (Figure 1.8). The
use of isotopically-labeled substrates in this investigation established that the
amino group at 02 and the vicinal pro-(3S) hydrogen are interchanged by PAM
reaction chemistry with retention of configuration at both migration termini, which
is an uncommon pathway for aminomutases (Table 1.2). The removal of the
hydrogen, which likely initiates the rearrangement, appears to be involved in the

rate determining step of catalysis (primary K.I.E z 2.0). Enzyme assays in which
D20 replaced H20 in the bulk solvent, indicated that there is significant, but

fleeting time-dependent exchange between solvent hydrogens and the a-
phenylalanine during the aminomutase reaction when the substrate was at
apparent saturation. This mechanism is patently different from the radical-
mediated lysine-2,3- and arginine-2,3-aminomutase mechanisms, wherein
exchange of hydrogen on the substrate with those of solvent is excluded.
Exploring the subtleties of the PAM mechanism is vital toward
understanding how this enzyme, involved in secondary metabolism, may direct
product selectivity and retain ammonia after vicinal isomerization compared to
the homologous and mechanistically-related phenylalanine ammonia lyases that
eliminates ammonia from the same substrate and provides compounds to
primary metabolic pathways. Furthermore, as B-peptides, comprised of B-amino
acids continue to become more conventional in the development of synthetic,

mimics of natural proteins antibiotics, PAM catalysis potentially provides an

30

alternative, tractable method toward the production of novel, stereospecific B-aryl

o-amino acid precursors for this application.

31

1.3 MATERIALS AND METHODS

1.3.1 Chemicals and Reagents

Deuterium gas, [ring, 2,3,3-2H31- and [3,3-2H2]-B-phenylalanines, and 020
(each at > 98% isotope enrichment) were purchased from Cambridge Isotope
Laboratories (Andover, MA). Ethyl benzoylacetate (90%) and [2H3]-ethyl acetate

(> 98% isotope enrichment) were purchased from Aldrich (St. Louis, MO). All
other reagents were purchased from or Sigma- Aldrich, unless otherwise noted,

and used without further purification.

EXPERIMENTAL PROCEDURES
1.3.2. NMR Spectra Measurements

All NMR measurements performed on a Varian UnityPlus500 and 1H
NMR spectra were acquired at 500 MHz. The proton-decoupled 2H-NMR

experiments were performed at 76.77 MHz.

1.3.3 GC/EI-MS Measurements

A gas chromatograph (model 6890M, Agilent, Palo Alto, CA) coupled to a
mass selective detector (model 5973 inerfE, Agilent) was used for the analysis of
derivatized products isolated from enzyme assays that were loaded onto a 5H8
60 column (0.25 mm inner diameter x 30 m, 0.25-um film thickness) mounted in
a GC oven. The MS conditions were set with an ion scan mode from 100 — 300
atomic mass units at 70 eV ionization voltage. The GC conditions were as

follows: column temperature was programmed from 70 °C (3 min hold) to 320 °C

32

 

at 10 °C/min and then a 3 min hold at 320 °C, splitless injection was selected,

and helium was used as the carrier gas (1.2 mL/min).

1.3.4 Heterologous Expression and Purification of PAM.

For the following procedures minimal growth media (MGM) (1 L contained
12.8 g N32HPO4'7H20, 3 g KH2PO4, 0.5 g NaCI, 1.0 g NH4CI, 2 mL 1 M MgSO4,
100 uL 1 M CaCIg, 10 mL 100x BME Vitamin solution, and 20 ml-20°/o D-glucose

solution) with ampicillin selection was employed instead of the previously used
LB to propagate the bacterial cells expressing the pam clone because a-
phenylalanine is an abundant ingredient in the LB media. The exogenous q-
phenylalanine was converted to B-phenylalanine in vivo during the over
expression of the pam gene in the transformed bacterial cells, and these amino
acids were co-extracted in considerable quantity (~0.3 mM) from the cells during
the clarification of the soluble enzyme fraction. The removal of these metabolites
prior to conducting kinetic analysis in the previous study required repetitive anion
exchange chromatography, with dialysis between reloading to remove sodium
chloride, followed by Ni-affinity purification chromatography, then final dialysis to
dilute 250 mM concentrations of imidazole to <1 pM. These manipulations
drastically reduced enzyme activity, making this an impractical approach for the

present investigation where significant quantities of biosynthetic q-phenylalanine
are required for detection by low-sensitivity 2H-NMR analysis. (ZS-phenylalanine

is a minor component in the MGM used, and accordingly, the levels of a- and [3-

amino acids in the soluble fraction of the bacterial extract were reduced by ~50-

33

fold. Therefore, the numerous purification steps to remove background amino

acids were avoided.

1.3.5 Optimization of PAM purification

In order to improve the purification of PAM using the Ni-NTA method the
polyhistidine tag was switched from the N-terminal to the C-terminal as follows.
By a cohesive-end PCR method, pam cDNA was subcloned from expression
vector pET1981His into pET14b to place the poly-His epitope tag at the C-
terminus (designated pET14-1981His). Preliminary purification of small-scale
preparations of PAM protein expressed from pET1981His and from pET14b
vector in E. coli BL21(DE3) indicated that the N-terminal-tagged protein bound to

Ni-affinity column matrix 8-fold better than the C-terminal-tagged protein (<10°/e

binding).100 The oligonucleotide primers for this sub cloning procedure have been

described previously.‘°° E. coli BL21(DE3) cells transformed with pET14-

1981His were grown for 16 h in 50 mL of MGM. These bacteria were used to

inoculate 6 L of MGM containing 100 pg/mL ampicillin. The cell cultures were
grown at 37 °C until 00600 = 0.6, over expression was induced by the addition of

1 mM of isopropyl-B-D-1thiogalactopyranoside, and the cultures were grown at
18 °C for 18 h. The remaining steps were conducted at 4 °C, unless otherwise
noted. The cells were harvested by centrifugation at 5,000 x g (20 min), diluted in
100 mL of resuspension buffer (50 mM potassium phosphate, pH 8.0), and lysed
by brief sonication (five 20-s bursts at 50% power (Misonix sonicator,

Farmingdale, NY)) followed by removal of cellular debris by centrifugation at

34

15,000 x g (30 min). Residual light membrane debris was removed by
centrifugation at 45,000 rpm (2 h). The clarified crude supernatant was partially
purified by anion exchange (primarily to remove small molecules) and HIS-Select
(Sigma-Aldrich) Ni-affinity gel chromatography (PAM eluted in 150 to 200 mM
imidazole). The fractions containing soluble PAM (~76 kDa) were combined (10

mL total), and subjected to iterative concentration-dilution cycles on size-
exclusion centrifugal filtration (Centriprep® Centrifugal Filter Units 30,000

MWCO, Millipore, Billerica, MA) to concentrate the protein solution to 3 mL and
dilute imidazole concentration to <1 uM. Low imidazole concentrations would not
confound the efficiency of our in situ amino acid derivatization chemistry nor
contaminate the GC column used routinely in these analyses. However, only
60% of the enzyme activity remained in the retentate compared to the activity
before removing the imidazole; no significant activity was found in the flow-
through, indicating that PAM either was being denatured in this step or was being
compacted unrecoverably into the filtration mesh during the >6 h centrifugation.
Therefore, to avoid further loss of enzyme activity while attempting to maximize
protein purity and dilute the imidazole concentration, the batch of protein at this
stage was used as the working stock in subsequent assays. The quantity of
concentrated enzyme (~50 ug/mL) was assessed by SDS-PAGE and Coomassie

Blue staining as described elsewhere, and this enzyme preparation was judged

100

to be ~70% pure. The modest protein purity of PAM obtained after two

chromatography steps was attributed to the abundant total protein isolated from

the large-scale (6 L) bacteria cell culture expressing pam. The protein milieu,

35

even after anion exchange chromatography, contained significant protein that

bound non-specifically to the affinity gel along with the Hise-tagged PAM. No

improvement in column selectivity was gained by varying the imidazole
concentration and increasing the salt concentrations in the wash solutions, as

suggested by the manufacturer, to reduce non specific binding.

1.3.6 General PAM Assay.

(28)-d-Phenylalanine (~500 pM) was added to 1 mL of 50 mM phosphate
buffer (pH 8.0) containing partially purified PAM (~1 pg) and incubated at 30 °C
for 2 h, and the production of (3R)-B-phenylalanine was evaluated. The assay
buffer was carefully basified (0.1 M sodium hydroxide) until pH 2 11, treated with
acetic anhydride (2 x 300 pL for 20 min between each addition), and the reaction
was quenched by acidifying to pH 2 with 1 M HCI. The N-acetyl amino acids were
extracted with ethyl acetate (3 x 1 mL), the organic fractions were combined, and
the carboxylic acids were methyl-esterified by diazomethane treatment. The
solvent was removed in vacuo, and the derivatized amino acid mixture was
dissolved in 300 pL ethyl acetate, and 1 pL of this solution was analyzed by

coupled GC/EI-MS.

1.3.7 Assessment of Hydrogen Exchange during the PAM Reaction.
PAM enzyme (1 ug/mL) was incubated with [ring,2,3,3-2H3]-(28)-d-
phenylalanine (250 (M) in 12 mL of 50 mM phosphate buffer (pH 8.0) at 30 °C,

and a 1-mL aliquot of sample was removed initially every 15 min up to 1 h, then

36

at various time intervals up to 50 h. A complementary experiment was conducted

wherein the water (H20) of a 50 mM phosphate buffer solution (2 mL, pH 8.0)

was removed in vacuo, the remaining phosphate salts were re-dissolved in 020
(2 mL), and the deuterated solvent was evaporated. The sample was again
diluted with 2 mL of D20, and to this volume was added PAM enzyme (1 pg)
solvated by 100 pL of protio-Iabeled buffer and 100 pL of a 5 mM solution (in
020) of unlabeled d-phenylalanine; this reaction was incubated to a single stop-

time of 17 h. The isolated q-phenylalanine isotopomers were converted to N-
acetyl methyl esters as described previously and subjected to mass

spectrometric analysis to assess the deuterium content.

1.3.8 Synthesis of N-Acetyl (2S,3R)- and (2R,38)-[2,3-2H21-Phenylalanine Methyl
Ester Racemate.

 

1. MeOH, H“ 0
o o 2. NH40Ac, Me0H //<NH o
/\ reflux, 5 h, 90%
O . . . E \ /
3. Acetyl Chloride, pyridine O
0 °C to 25 °C, 18 h, 95%
Ethylbenzoylacetate (Z)-Methyl-3-acetamido-3-phenylacrylate
i O

NH 0 NH 00

. o ;
02 (gas), 30 psr : ' 0/ + . 0/
1o % wt, Pd/C, MeOH: 98% o "H H ”D

(28, 3R)-, (2R, 3S)-N-acetyl-B-methyl ester racemate

 

Scheme 1.12 Synthesis of (2S, 3R)-, (2R, 3S)-N-acetyl-j3-methyl ester racemate.

37

The following synthesis procedure (Figure 1.12) is an adaptation of an
established method.111 To a stirred solution of ethyl benzoylacetate (57 mmol, 11
mL) dissolved in methanol was added concentrated sulfuric acid solution (0.5
mL). After 20 h, the solvent was evaporated and the resulting oil was partitioned
between dichloromethane and water. The organic layer was collected and
evaporated to yield methyl benzoylacetate, and used in the next step without
further purification. The oil was dissolved in methanol (150 mL) and excess
ammonium acetate (20 g) was added, and the solution was refluxed for 5 h. The
reaction was cooled and the resulting crystals were removed by vacuum filtration.
The solvent was then evaporated to yield the desired (Z)-methyl-3-amino-3-
phenylacrylate enamine as an oil, which was purified over silica using 9:1

hexanes/ethyl acetate, v/v. A yield of 90% was obtained based on the ethyl

benzoylacetate (9.0 g, 51 mmol). 1H-NMR (300 MHz, CDCI3): 6 3.70 (00H3),

4.98 (HC=C-), 7.48-7.60 (phenyl-H). GC/El-MS: m/z 177 [W], 146 [M - OCH3]+.

1.3.10 Synthesis of (Z)-methyl -3-acetamido-3-phenylacry/ate

To the purified enamine (11.3 mmol, 2 mL) in dry THF (50 mL) was added
freshly distilled pyridine (10 mL), and the solution was cooled to 0 °C. Freshly
distilled acetyl chloride (84.8 mmol, 7.5 eq., 6 mL) was added drop wise, and the
reaction was warmed to room temperature and stirred overnight. Reaction
progress was monitored by TLC, and additional acetyl chloride was added drop
wise to complete the reaction. The sample was quenched by dilution with brine

(50 mL) and extracted with chloroform (3 x 25 mL). The solvent was evaporated

38

and the N-acetyl enamine methyl ester was purified by silica gel (85:15
hexanes/ethyl acetate, (v/v)). The solvent was evaporated and the desired (2)-

methyl-3-acetamido-3-phenylacrylate was obtained quantitatively. 95% yield (2.3
g, 11 mmol). 1H-NMR (300 MHz, 00013): 5 = 2.14 (s, CH30=0), 3.70 (s, 00H3),

4.98 (s, C=CH), 7.48-7.80 (phenyl-H). GC/El-MS: m/z 219 [M*], 177 [111+ -

CH30H], 160 [M - 0020H3 or M+ - HNC(0)CH3]+.

1.3.11 Synthesis of Dideuterio N-acetyl-(28,3R)-, and -(2R,3S)-,8-Phenylalanine
Methyl Esters

To the (Z)-acrylate (6.0 mmol, 1.3 9) dissolved in methanol (30 mL) in a
Parr flask was added 10% w/w Pd/C (25 mg). The chamber was pressurized with

deuterium gas at 30 psi to stereospecifically reduce the substrate by syn-
addition. The reaction was shaken on a Parr Hydrogenator® for 24 h then filtered

through Celite overlaying a short (~2 cm) column of silica to remove the catalyst.
The methanol was evaporated to provide a racemic mixture of dideuterio N-
acetyl-(28,3R)- and (2R,3S)-(3-phenylalanine methyl esters ( > 98 % yield, 1.2 g).

Proton-decoupled 2H-NMR (79.77 MHz, ethyl acetate): 6 = 2.82 (2H at C2), 5.41
(2H at C3). GC/El-MS: m/z 223 [W], 180 (~1” - CH30 (0)]. 100%), 149 [M+ -
HC(D)C(0)OCH3], 20%). This compound was used to evaluate the

stereochemistry of the hydrogen migration from C3 to 02 of the phenylpropanoid

substrate in the PAM reaction. Since authentic B-phenylalanine specifically

deuterium-labeled at 02 was not available, this synthetically-derived [2H]-labeled

39

compound was also used to assess the extent of D- for H-exchange at 02 under
the same basic and acidic conditions during the derivatization of the a- and [3-

phenylalanines to their N-acetyl methyl esters. Approximately 500 pM of the
dideuterio N-acetyl amino acid ester was suspended in 1 ml of H20 which was

adjusted to pH 11 (0.1 M Na0H) in a 10 mL glass screw-capped tube and
shaken vigorously (Vortex) for 1 min. The solution was then acidified (pH 2) with
dropwise addition of 1 M HCI, and shaken for 1 minute. The suspension was
extracted with ethyl acetate (300 ,uL), and the organic fraction was dried by
' passage through a column of sodium sulfate powder. A 1 pL aliquot of the eluant
was analyzed by GC-EI/MS, and the diagnostic ion fragments revealed that the
isotopic abundance of the base/acid-treated analyte was identical to that of the
starting material, which was at 98% deuterium isotope enrichment, indicating that

no H/D exchange occurred at 02 under the conditions outlined above.

1.3.12 Evaluation of the Stereochemistry of the Hydrogen Migration in the PAM
Reaction.

1H-NMR analysis of unlabeled (28)-q- and (Sm-B-phenylalanines, at 200
pM each, dissolved in perdeuterio-ethyl acetate, was conducted to assess if the

relative chemical shifts of the protons at 03 of the d-isomer and those at C2 of

the B-isomer were well-resolved (Figure 1.8.4). Once the peak resolution was
observed to be sufficient at 0.24 ppm, [3,3-2H2]-(2S)-q-phenylalanine (5 mM) was

incubated with PAM (~ 50 pg) in each of two 1 mL samples (10 pmol substrate

total) in phosphate buffer (pH 8.0) at 30 °C. The reaction progress and deuterium

4O

distribution in the product were tracked by GC/MS analysis at regular intervals
after the start of the reaction by removing aliquots (1 mL) from each sample, and
the amino acids were derivatized as described earlier. Under these assay
conditions, where 50-fold more protein was used than in a routine assay, the
reaction was halfway to equilibrium at 6 h. Thus, the reaction was terminated at
10 h by careful basification to pH 10 (0.1 M sodium hydroxide solution), and the
isotopomeric amino acids were derivatized as before, except that 20 pmol acetic

acid was used. An estimated 830 pg of product (5 pmol, ~50 mol% conversion of
substrate to product) was made, which was sufficient for analysis by 2H NMR.

The biosynthesized dideuterio-B-phenylalanine isotopomer was present at ~30%
(~330 pg, 1.5 pmol) compared to the monodeuterio-B-isotopomer as judged by

GC/EI-MS analysis. Diagnostic fragment ions were found at m/z 179

[PhCaD(NH)CBH2CO2CH3+, 100%], 180 [PhCaD(NH)CBDH0020H3+, 28%], and

149 ([PhC(D)=NHAc+], 20%). The dried mixture of derivatized deuterium-labeled

q- and B-amino acids extracted from the aqueous assay solution was dissolved
in 300 ,uL ethyl acetate and analyzed by proton-decoupled 2H-NMR, with

overnight scanning. The chemical shifts corresponding to the deuterium of the

labeled B-phenylalanine in the sample were compared to those of the authentic
(28, 3R)- and (2R, 3S)-[2, 3-2H2]-N-acetyl B-phenylalanine methyl ester racemate
dissolved in ethyl acetate to assess the stereochemistry at C2 of the
biosynthetically-derived product. For reference, the [3,3-2H21-(28)-q-

phenylalanine substrate (5 mM) was isolated from 1 mL of assay buffer (50 mM

41

phosphate buffer, pH 8.0) without PAM enzyme after a 12 h incubation at 30 °C.

The substrate was derivatized to its N-acetyl methyl ester by described methods,

dissolved in 300 pL ethyl acetate and analyzed by 2H NMR.

1.3.13 Assessment of the Kinetic Isotope Effect.
PAM and an equal mixture of [ring, 2,3,3-2H3] - and unlabeled-(28)-q-

phenylalanine (500 pM of each) were incubated for 3 h until ~ 0.2% conversion of
q- to B-phenylalanine (2.3 nmol) was observed under standard assay conditions.
The amino acids were converted to their N-acetyl methyl esters as described
previously, and the relative amounts of product derived from labeled and
unlabeled substrate were compared. The isotopomers generated diagnostic base

peak fragment ions in the mass spectrometer at m/z 178 (unlabeled product)
(Table 1.1A), and at 185 and 186 (07 and Dg-labeled product, respectively)

(Table 1.18); the deuterium originating at C3 in the labeled substrate is partially
lost during its transfer to C2 in the isomerization reaction. The ratio of the peak

area for ion m/z178 to the combined areas of ions m/z1‘85 and 186 was used to

calculate the kinetic isotope effect on Vmax/KM in this competitive assay.112

1.3.14 Measurement of the Equilibrium Constant

To assess the equilibrium between product and substrate in the PAM
reaction at 30 °C, car-phenylalanine at 100 pM was added to 10 mL of PAM (at
~25 pg/mL instead of 1 pg/mL used for routine assays) in 50 mM phosphate

buffer (pH 8.0). Aliquots (1 mL) were withdrawn from the reaction at various time

42

points between 0.25-50 h. The reaction in each aliquot was stopped by careful
addition of 0.1 M sodium hydroxide, and the amino acids were derivatized and
analyzed by GC/EI-MS, as described above. The relative amounts of product and
reactant at equilibrium were determined by linear regression analysis of the area
of the base peak fragment ion of the derivatized d- or B-phenylalanines (m/z162
and 178, respectively). Peak area was converted to concentration-of-product (or -
substrate) by solving the corresponding linear equation, which was derived by
plotting the area of the signature base peak ion, produced by the corresponding
authentic N-acetyI-amino acid methyl ester standard, against the concentration of

each standard in a dilution series ranging from 0 - 30 pM, at 5 pM intervals.

43

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Michel, 0.; Albracht, S. P. J.; Buckel, W., Adenosylcobalamin and
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53

CHAPTER 2
Analysis of the Substrate Specificity of Phenylalanine Aminomutase (PAM)

2.1 INTRODUCTION
B-Amina Acid-Based Natural Product Pharmaceuticals

B-amino acids in general are constituents of several antibiotics and
pharmaceutically-active natural products, such as januvia, blasticidin and taxol.

Januvia (sitagliptin phosphate) (Figure 2.1) is a first in a class of new oral
medication which has been approved by the US. Food and Drug Administration
for management of Type 2 diabetes.‘ It is in the category of drugs called (DPP-4)
dipeptidyI-peptidase 4 inhibitors. OPP-4 is responsible for breaking down the
proteins that stimulate insulin producing cells after a meal. If DPP-4 is inhibited,
then the proteins can activate the release of insulin for a longer period of time,
thereby lowering the glucose level in the blood. It is prescribed for Type 2
diabetes only.

Blasticidin S (Figure 2.4) is an antibiotic used to prevent the growth of both
eukaryotic and prokaryotic cells, and functions by inhibiting peptide bond
formation with the ribosome.2

Taxol (Figure 2.1) is a taxane diterpenoids”13 used to treat several cancers

14-31

including ovarian, metastatic breast (anthracycline-resistant), non-small cell

lung cancer, small-cell lung cancer, squamous cancers of the head and neck and

32-34 35-38

AIDS-related Kaposi’s sarcoma and some Ieukemias. Cancer is the

second cause of mortality in the USA and is responsible for one in four deaths.

54

The annual sale of Taxol® in 2006 reached 3 billion USA dollars. It is currently

under investigation for the potential treatment of several other diseases including

39 40-42 43-51

polycystic kidney diseases, coronary retenosis and Alzheimer’s.
Although Taxol is a promising drug, there remain the challenges of drug
resistance that is commonly observed with most antibiotics. Therefore, the
development of alternative or next generation analogues of an existing drug
becomes paramount. Biocatalysis offers the most flexible synthetic method of
accessing second generation Taxols, which requires the optimization of selected
enzymes found in the biosynthetic pathway of paclitaxel.

Other drugs that contain B-amino acids in their structures include the

amino peptidase inhibitor bestatin52 from Streptomyces globisporous, the anti-

53'58 myomycin, viomycin,59 roseathricin, geomycin

type II diabetes drug januvia,
and tuberactinomycin.59 Single B-aryl-B-alanines have been shown to have anti-

epileptogenesis activity,60 while other amino acids have been used as building

blocks toward the synthesis of complex bioactive molecules, including B-lactams

and B—peptides as mimics of a-peptide hormones and as antimicrobial

compounds.61

55

 

F

Januvia

Flgure 2.1 Structure of taxol and januvia

Current sources of Paclitaxel
A semi-synthetic method was developed as a viable approach to address

6.6268

the supply issue. This process involves extracting an advanced Taxol

precursor, baccatin lll, from the leaves of the European Yew Taxus baccatta, and
from T. wa/Iichiana or T. cuspidata. This precursor is protected at 07 position and
coupled with an N-benzoyl phenylisoserine side chain precursor, as a
synthesized B-lactam (Scheme 2.2). This is a relatively efficient process wherein

a kilogram of either 10-deacetylbaccatin III or baccatin Ill can be converted into

about 0.6 to 0.7 kg Taxol.”69

56

N
0

O
2 Fri SiO/ “Q
° 9”“ ‘ ’3 J: (1 *0
: mg 0
NH 0 .

B- -Iactam 0

 

(i) a

(3)70 (ii) ' ‘
2:273 Paclitaxel (Taxol) )=O

R1, R2 = H: 10-Deacetylbaccatin lll
R1 = H, R2 = Ac: Baccatin lll
R1 = SiEt3, R2 = Ac :7-TES-baccatin ll|

 

 

Scheme 2.2 Coupling of baccatin III with a B-lactam produces Taxol in two steps; (i) NaH, THF,
B-lactam and (ii) HF, pyridine. The B-lactam was synthesized in seven steps.

Recently, Bristol-Myers Squibb collaborated with Phyton to develop a

plant tissue culture technique Le. a plant cell fermentation (PCF) technology”82

to produce paclitaxel. In this biosynthetic process, caIIi cultures of a specific
Taxus cell line are propagated in aqueous medium in large fermentation tanks.
The cell cultures are treated with methyl jasmonate to induce paclitaxel
production, and paclitaxel is then extracted directly from cell cultures. The Taxus

plant cell culture fermentation improved the sustainability and titers of the

80,83

paclitaxel supply. While the plant cell culture technique offers a sustainable

resource, it does not offer the flexibility to produce modified paclitaxels with

greater efficacy in terms of solubility, cytotoxicity, or targeted delivery, hence the

57

need to continue to explore better methods of synthesis by optimizing some

enzymatic steps involved in the biosynthesis of this important drug.

Taxol Biosynthesis

In Taxus plants paclitaxel biosynthesisis initiated from the metabolism of

methylerythritol phosphate (MEP)84 pathway, which stems from the

85-91

glyceraldehydes and pyruvate. MEP is isomerized to isopentyl diphosphate

92-94

and dimethylallyl phosphate. Three lPP and two DMAP molecules condense

to form the diterpenoid progenitor geranylgeranyl diphosphate (GGPP)95 by

action of a geranylgeranyl diphosphate synthase (GGPPS). Taxadiene synthase

(TS) then cyclizes GGPP to form the tricyclic taxa-4(20),11(12)-diene. Eight

96-1 22

cytochrome P450 mediated hydroxylation, three acyl/aroyl CoA dependent

transferases [taxadiene-Sd-ol-O-acetyl transferase (TAT), 211-0-
benzoyltransferase (TBT) and 10-deacetylbaccatin-10-acetyl transferase

(DBAT)], and a presumed rearrangement of an acetoxy oxirane to an oxetane

100

formation) (Scheme 2.3) results in the formation of baccatin Ill.

The biosynthesis and attachment of the C13 side chain for the baccatin Ill
moiety involves 5 steps. First, is the conversion of (S)-q-phenylalanine to (R)-B-

phenylalanine by a phenylalanine aminomutase, secondly the B-amino acid is

123-128

coupled to coenzyme A by a putative ligase, and the third step involves the

transfer of the phenylpropanoid to the baccatin Ill core by a

phenylpropanoyltransferase to form N-debenzoyl-2'-deoxypaclitaxel.129

58

Hydroxylation at the 2' position by a P450 is the penultimate step followed by

benzoylation by a N-benzoyltransferase utilizing benzoyl CoA as a

substrate.13°'131

59

 

New

20H 0

.. GGPPS
”mot-1.9“ \ New >~ (mm—s. -—

90 °“
H ’I :9 ."

 

 

 

...__ /"ZT
Grime“ 02200 4&0

AcO

 

 

 

 

Scheme 2.3 Biosynthesis of paclitaxel. The proposed mechanism for the oxetane ring formation
is shown in the inset.

Strategies for synthesizing [3 amino acids
Several strategies have been used for the synthesis of various substituted

B-amino-B-arylpropionic acids that include multi-step processes, and one pct

60

procedures such as the Knoevenagel condensation of benzaldehyde and

132

malonic acid in the presence of an amine source (e.g. NH4OAC). Recently,

Node and co-workers reported a method of synthesizing B-amino acids through a
Michael addition of a chiral amine to d,B- unsaturated esters, and the
stereoselectivity was inverted by changing the solvent from diethyl ether to

tetrahydrofuran (THF) when o,B-esters having an aromatic ring at the B-position
were employed”:3 Good yields of B-amino acids with an enantiomeric excess of

82-99% have been reported, but the starting materials are protected as N-

methoxycarbamates and the reaction is catalyzed and stereocontrolled by chiral
pyrrolidines derivatives, which are difficult to access.177 Recently, Parra reported
a new approach to B-amino acids synthesis, which is based on the addition of
dianions of carboxylic acids to isocyanates under acidic conditions.134 The

dianions are generated using lithium cyclohexylisopropylamide as base at -78 °C.

All the chemical methods reported so far use solvents which are toxic to
the environment. However, there are presently no reports on biosynthetic
strategies toward the production of asymmetric B-aryl-B-alanines from the
corresponding d-isomers which are greener in nature. Tyrosine aminomutase
(TAM) from Streptomyces globisporous is involved in the biosynthesis of

enediyne C-1027 anti-tumor agent, TAM also has broad substrate specificity and
is able to accept ring-chlorinated tyrosines as substrates.135'136

The Taxus phenylalanine aminomutase (PAM) (Scheme 2.11) provides a

potential alternative route toward scalable production of novel B-amino acids as

61

 

drug intermediates. This isomerase shows high amino acid sequence homology

(56% similarity) to a family of ammonia lyases that have broad substrate

137

specificities with derivatives of aromatic amino acids. Despite the wealth of

information on the substrate specificity of the MIO-based reactions of the
ammonia lyase family, there remains a paucity of investigation on the scope of
the substrate specificity of the PAM enzyme. The potential of the mutase enzyme
to biocatalyze the production of novel B-amino acids prompted a survey of
several commercially-available S-a-amino arylpropanoic acids as substrates to

examine specificity.

62

 

 

 

 

 

 

 

 

 

C>
Howe Amino migration Chlorination 8Hydroxylatron
Cl
6)
H3N HO H3N HO PC P H3N \‘HO
:Wo Adenylation HO 0 Ad = HO ‘ S,PCP
HO
Cl
Condensation

V

102?:CH3

“Elf“
H c N
(3)2 10.4.30 l
OHH
CH3
OH Cl 0

 

H2N vH

Scheme 2.4 Biosynthesis of 01027 an antibiotic and antitumor agent isolated from
Streptomyces globisporous. The first step of one of the biosynthetic branches to C-1027 is
catalyzed by tyrosine aminomutase which converts o-tyrosine to B-tyrosine (inset)

63

2.2 RESULTS AND DISCUSSION
2.2.2 RESULTS
2.2.2.1 Substrate specificity evaluation

Enzyme assays were conducted with 25-50 ug/mL (> 70% pure) PAM of enzyme
at 31 °C, over 3 h and the productive substrates are shown in Table 2.1. For poor
substrates such as 4'-t-butyI-a-phenylalanine and (28)-styrylalanine the reactions
were incubated with 500 ug/mL enzyme for 3 h in order to increase the product

yield at steady-state.

Table 2.1 Kinetic Parameters of PAM with various substrates. The Vrel is the ratio of VMAx for a
particular substrate and VMAx of phenylalanine. The standard deviations are indicated in

parenthesis.

 

 

 

 

 

 

 

 

 

 

 

 

SUBSTRATE 0“ vMAX VMAx/KM
x/YKO KM (PM) -1 - -1 Vrel
NH2 (nmol-h ) (nmol0h OpM )
DA 75 (17) 180 (10) 2.4 (0.6) 95 (7)
1 F
S "‘44,
g 35(8) 26(1) 0.74 (0.17) 14 (1)
2
410 (46) 48(1) 0.12 (0.01) 25(1)
3 H3CO
Fl \ 330 (65) 14(1) 0.042 (0.01) 7.4 (<1)
4
DA 160 (39) 4.4 (0.1) 0.028 (0.007) 2.3 (0.1)
5H30
©/\ 97 (6) 1.9 (0.1) 0.020 (0.002) 1.0 (0.1)
6

 

64

 

Table 2.1 (continued)

 

VMAX

' VMAx/ KM

 

 

 

 

 

 

 

\
12 ><©/

 

 

 

 

SUBSTRATE 0” v.
X/YKO KM (“Mi -1 -1 -1 '3
NH2 (nmol-h ) (nmol-h ouM )
HaC \
944 8.9 (0.75)
U (100) 17 (0.15) 0.018 (0.0012)
7
F \ 0.0057
0 560 (60) 3.2 (0.30) 4 1'7 (0'15)
(5.2 x 10 )
8
\ 2 2 x 10'3
Ii): 50 (5) 0.11 (0.01) ' 4 (333:)
9 CH3 (3.0x10' ) '
{Dj/\ 410 50 003 0001 7'3X105 “016
\ ( ) ' ( ' ) (1 0x105 (0.001)
10 -
Q/V\ 0 078 1 0"").4 o 041
780(71)
1 1 (0'00” (1 .0x10'5) (0°02)
3 -5
~10 <0.01 «10 «0.001

 

B-amino acids were commercially-available for use as reference products,
in derivatized form, except styryl-B-alanine which was synthesized as described
below. Given the strict stereospecificity of PAM for S-phenylalanine and
stereoselectivity for the production of R-B-isomer it was conceived that all of the
unnatural B-amino acids made biosynthetically were also R. A chiral column

however was used to establish the stereochemistry of the B-products by GC/MS

analysis.

65

 

2.2.2 Stereochemistry of the fi-amino acids

-1 -1
The catalytic efficiency at ~0.02 nmol-h opM of PAM for the 3'- and 4'-

methyI-o-phenylalanine substrate (7 and 5, respectively) is nearly equal to the

value for the natural substrate (6) (Table 2.1). However, noticeably higher Vrel

values of 8.9 and 2.3 for the 7 and 5, respectively, were observed (Table 2.1),

-1 -1
while a Vrel at 0.05 and catalytic efficiency at 0.0022 nmol-h opM for the

isomerization of the 2'-methyl regioisomer (9) was substantially lower. This
apparent rate decrease is likely related to steric interactions of the ortho-methyl
group (in place of H at one of the two ortho-carbons) (Figure 2.6) with active site
residues, or the MIO, resulting in non-productive binding.

The Vmax of PAM for 4'-t-butyl-a-phenylalanine (12) was slower (Vrel <

0.001) than 6; however, the modest isomerization of 12 reveals that the PAM
active Site can occupy a relatively bulky, aliphatic substituent at the para-position
of the substrate (Figure 2.7). However, the nine —C—C—H bond extensions of
the t-butyl group likely places 12 near the steric limits of the active Site compared

to the smaller 4'-methyl homolog 5.

66

MIO

/\N.\(\

ortho H :1" (+3
- \NH3

‘ H
0009

Figure 2.6 The aromatic ring attack on the terminal double of the catalytic MIO could be blocked
by the presence of a methyl group at the ortho- position of the substrate L-phenylalanine leading
to lower reactivity compared with the natural substrate.

Phenylalanine analogs bearing electron-withdrawing or electron-donating
groups on the aromatic ring were used to investigate the effects of electron

induction on PAM at steady-state. The catalytic efficiency of PAM catalysis for

the 4'-fluoro-o-regioisomer (1) at 2.4 nmoI-h'u'uM.1 was greatest of all of the

substrates tested. The 2'-fluoro isomer (4) was less efficiently catalyzed (0.027
nmoloh'1-uM'1) by ~60-fold compared to 1, while the 3'-fluoro isomer (8) was even

less efficiently isomerized (0.0057 nmoloh'1ouM) by ~400-fold (Table 2.1). The
turnover of each fluorinated substrate was greater than for phenylalanine. In

general an electron-withdrawing flouro on the ring of the substrate may increase
the acidity of the [3.4 of the substrate (Figure 2.8), thus increasing the overall

reaction rate.

67

 

 

--------- SH" ,1
\<:80 / {3.39 lle383
Leu79 ( .

4.58 \ 3:3

'. ; ,2’295 fl,
\ . ‘\ ‘ I ’ ,,,,
x \ \\1 __ fiv’
MI “11‘ H O 3'56 ; Val386
Leu 191 2,50 *
O Glu414
I . HN

OH3N Ar9283

 

Leu194

Asn195

 

 

 

 

Figure 2.7 Shows a model of phenylalanine anchored in the active site of PAM with the phenyl
ring interacting with hydrophobic residues. The approximate interaction distances between the
substrate and the active site residues are indicated (A).

The 4'-methoxy-d-phenylalanine substrate (3) also demonstrated greater
efficiency (0.12 nmol-h'1-pM'1) and superior turnover (V 9' = 25) compared to 6

(Table 2.1). The 3'-methoxy regioisomer was surprisingly not a productive
substrate, especially considering that the 3'-methyl substrate was converted to its

B-isomer nearly 4 times faster than the 4'-methyl isomer. Perhaps steric

68

interactions are more significant with the larger -OCH3 group, thus precluding

proper active site binding.
Heteroaryl-B-phenylalanines, furan-2-yl- (10) and thioen-2-yl-S-o-
phenylalanines (2), were tested as substrates of PAM at steady state, and 2 was

found to produce B-amino acid product much faster (Vrel = 14) than 10 (Vrel =

0.02) (Table 2.1). The efficiency of PAM with 2 was second only to that of the 4'-
fluoro species 1, and was also isomerized ~40 times more efficiently than 6. (S)-

Styrylalanine (11) was found to be a productive substrate of PAM with a Vrel =

0.04 compared to 6 (Table 2.1); although, the catalytic efficiency (VMAX/KM z

-1 -1
0.0001 nmol-h ouM ) was comparatively much lower. Notably, PAM was unable

to convert the saturated styrylalanine analog ((S)-2-amino-5-phenylpentanoic
acid) to its B-isomer, suggesting that an extended conjugated allyl Tr-system next
to the q-carbon, bearing the migrating amino group, is required for isomerization.

Allylglycine was found to be a non-productive substrate of PAM based on
the expected retention time and fragmentation pattern of the B-N-acetyl
allylglycine methyl ester standard. The lack of conversion could be due to
improper binding of the substrate within the active, which probably confirms to
aromatic amino acids by interaction with the hydrophobic leucine and isoleucine
residues that are conserved among enzymes belonging to the MIO family (Figure
2.7). The other possibility could be that after the elimination of the amine a
conjugated allyl system iS formed and the rebounding amine could add to the y-

or 6- positions of the resulting polyunsaturated pentenoic acid. 80, instead of the

69

 

expected B-allylglycine other compounds could be formed such trans-4-
aminopent-2-enoic acid or trans-5-aminopent-3-enoic acid (Figure 2.8). Both
compounds were synthesized, and no signal corresponding to the N-acetyl
methyl ester derivatives of the amino acids was observed by GC/MS. These
negative results suggested that the phenyl ring may be required for binding to the

active site (Figure 2.7).

 

COOH NH2
/\/4 M
M00011 <— / NH2 —-> COOH
NH2 a- a||y|g|ycine (E )-4-aminopent-2-enoic acid

13- allylglycine

(E)-5—aminopent-3-enoic acid

 

 

 

Figure 2.8 Shows the possible products of the q-allylglycine-PAM reaction. The loss of the 0-
amino group could lead to an extended conjugated system which may result in the addition on the
y and 5 positions to form the 4- or 5- amino-trans-pent-3-enoic acids. However both compounds
were not identifiable by GC/MS/El analysis.

2.2.3. In vivo Screening of Amino acids
Phenylalanine analogues (1 mM) were fed to a 50 mL culture of
transformed BL21 (DE3)pLysS over expressing the PAM enzyme. The amino

acids were derivatized in both the cell pellets and the supernatant (Figure 2.9).

70

 

4MS

Mol °/o B—phe/g dry weight/Titre

Th5
ZFS FUS 4MOC

3FC 3F4MC ZFC ThC 4MOS

 

 

 

Flgure 2.9 A profile of in vivo synthesis of a mixture of [3- amino acids. The a-amino acids were
incubated with E. coli transfected with a pam plasmid expressing phenylalanine aminomutase

(PAM), at 37 °C 16 h. The PAM expression was induced with IPTG at an 00600 ~ 0.6. The
products were assayed in both (C) cells and supernatant (S). 3M is 3'-methyl-B-phenylalanine;
4M is 4'-methyl-B-phenylalanine; 2M is 2'-methyl-B-phenylalanine; 3F is 3'-flouro-B-phenylalanine;
4F is 4'-flouro-B-phenylalanine; 2F is 2'-flouro-B-phenylalanine; Fu is 2'-furanyl-B-alanine; Th is 2'-
thienyl-B-alanine and 4M0 is 4'-methoxy-B-phenylalanine.

2.2.2.4 Biocatalysis

The results Shown above (Table 2.1) provides a synthetic technique for
accessing B-amino acids both in vivo and in vitro with less toxic waste to the
environment compared to the chemical synthesis methods. The general scheme

for the biocatalytic synthesis of aryl-B-alanines is shown in figure 1.9.

71

 

 

G) \ Y
°H .H" 5 °H 9H3 3 Ar= |// (x 77
' PAM ‘
Ar)3\r2%°° 4%;er x

@1113 H’ x = F, CH3, CH3O, (CH3)3C Y = o, s

 

 

 

 

Figure 2.10 General scheme for the synthesis of various stereospecific beta amino acids.

Second generation paclitaxel can be access in a one pot reaction both in vivo
and in vitro by incubating selected recombinant enzymes (acyl transferases,
PAM and ligase) expressed in E. coli with some metabolites found in the
biosynthetic pathway of paclitaxel (Figure 2.11). Most of these enzymes have
already been demonstrated to be promiscous just like PAM. The various groups

which can be introduced on to the baccatin lll core are shown in Figure 2.11.

72

 

 

O

0
W051 (:1)K 0“
NHZ Benzoic acid

(yr-phenylalanine CoAS
Ligase
PAM

HzN 0

WW (DES-00A

B phenylalanine Benzoyl CoA
COASH

”2'9 0
(:("V‘Ls-CoA

phenylisoserine CoA

it

000

H
Hot-K236" O

°7=o

 

 

 

 

N-Debenzoyltaxol transferase

baccatin Ill
HO O OH

H2N 0 HO O OH
O‘
(>/\/‘L H% 62.0):0 OP450_.e©/\/1LO‘OO“O
()be hyroxylase 0H Hde-I O)=O

N-debenzoyl-2'-deoxytaxol N-debenzoyltaxol

Scheme 2.11 Biosynthesis of taxol from a-phenylalanine which is first converted to
B-phenylalanine (boxed) by the enzyme phenylalanine aminomutase(PAM).

73

 

 

 

\

R1: ’ Q/ _

Q x X-S,NandO
G

G = F, MeO, CH3, (CH3)3C

\

R2= . Q/ -

Q X X-S,NandO
G

G = F, MeO, CH3, N02, (CH3)3C

\

R3= . Q/ -

Q X X-S,NandO
G

G = F, MeO, CH3, (CH3)3C

 

 

 

 

R4 = CH3, CH3CH2

Figure 2.11 The proposed biocatalysis of of Taxol involving various enzymes found in the
biosynthetic pathway of taxol.

2.2.2.5 Mass spectral analysis of biosynthetic products generated by PAM
catalysis

Tandem gas chromatography/mass spectrometry was used to separate
the N—acetyl (or N-benzoyl) methyl ester derivatives of B-amino acids isolated
from assays in which corresponding B-amino acids were incubated with PAM. In
each pairing, mass spectra of the authentic standards (top panel) and of the
corresponding biosynthetic sample (bottom panel) are shown (Figures 2.13.1-
12). The retention times of the standards and biosynthetic products on GC were
identical. 1, N-benzoyl 4'-fluoro-B-phenylalanine methyl ester; 2, N--benzoyl-B-(2-

thienyl)-B-alanine methyl ester; 3, N-benzoyl 4'-methoxy-B-phenylalanine methyl

74

 

ester; 4, N-benzoyl 2'-fluoro-B-phenylalanine methyl ester; 5, N-acetyl 4'-methyl-
B-phenylalanine methyl ester; 6, N-acetyl B-phenylalanine methyl ester; 7, N-
acetyl 2'-methyl-B-phenylalanine methyl ester; 8, N-benzoyl 3'-fluoro-B-
phenylalanine methyl ester; 9, N-acetyl 3'-methyl-j3-phenylalanine methyl ester;
10, N-benzoyl-B-(Z-furanyl)-j3-alanine methyl ester; 11, N-acetyl styryl-B-alanine

methyl ester; 12, N-acetyl 4'-t-butyl-B-phenylalanine methyl ester.

75

1.0-

0.8 ~

0.6 -

0.4 -

0.2 4

51

 

 

105

 

122

 

 

149
‘64 228240
l .l. 1 .1 I 212.1 27° 301

 

1.0-

0.8 a

0.6 d

0.4 ~

0.2 ‘

 

4 l 1 .
198

105

122

149164 228240
212 270
l 1| I1 1 . .1 1 301

I 1
l I I l I

 

 

 

 

 

100 150 200 250 300
m/z

Flgure 2.12.1 N-benzoyl 4'-fluoro-B-phenylalanine methyl ester

76

1.0 n

 

 

 

 

 

 

 

 

 

 

 

 

0.8 T
0.6 -
(14 3 105
0.2 - 77
110
152 215
51 137 200 258 289
L 4917 1 l 111 11 1 ll 1 1
1.0" 105 184
0.8 4
77
0.6 a
0.4 -
51
0.2 - 110
I 137 152 2162 8
97 l 111 200
l 111' 11': (I1 [258 2519‘
50 1 00 1 50 200 250 300

Figure 2.12.2 B-(2'-thienyl)-B-alanine-N-benzoyl methyl ester

77

 

 

 

 

 

 

 

 

 

 

1 o q 208 IN
105 E
0.8 -
0.6 4
77
0.4 -1
134
0.2 -
50 1 1 24°
6 L 252 313
LL‘» L l L J] 1 L l 1 l 1 L 1 1
1.0 . 298
1
105 1
0.8 " g 1
l |
1
1 1
0.6 ~ 1
1 1
7,7 1
0.4 1 l 1 1
1 1 '
‘ 1 134
1 l .
0.2 - 1 1 |
1 . i
50 .‘ 1 1 1 . ': 240
l l ! 161 1 1 I
1 l 1 1 ( | ( 1 $ 252 313
1.1 .1 1.; I ,1 A (‘1 l. u 1 ' 1 '1 I 1
50 100 150 . 200 250 300
m/z

Figure 2.12.3 N-benzoyl 4'-methoxy-B-phenylalanine methyl ester

78

 

 

 

 

 

 

 

 

 

 

 

 

 

1.0 _ 105 195 Ph
0.8 T
0.6 a
77
0.4 '-
0.2 1 12 228
51 149 164
240
.. 1111111111L .1130 212 213
1.0 - 11’5 ‘96
0.8 3
77
0.6 '1
0.4 a
02 122
' 51 8
119 1
240 2
11' 1. I11“ I ”III "I 111 I To 1?) l 212 J]; I 1 F
50 100 150 200 250

m/z

Flgure 2.12.4 N-benzoyl 2'-fluoro-B-phenylalanine methyl ester

79

 

 

1.0] 192

0.8 *

 

0.6 -

0.4 ~

0.2 . 133

 

 

 

 

. ll ‘15 215

192

 

1.0 n
0.8 a

0.6 4 120
0.4 u

_ . 133
02 91

145 162

65 77 105 11 LL 11: 235

50 100 150 200 250
m/z

 

 

 

 

Flgure 2.12.5 N-acetyl 4'-methyI-B-phenylalanine methyl ester

80

100-

20-

106

 

119

131

178

 

 

 

100-

301

 

 

 

106

 

 

.11

.1 1

119
131

 

,1. 1...,

148

1 1111

161

111
l

 

 

100

Flgure 2.12.6 N-acetyl B-phenylalanine methyl ester

120

140

160

m/z

81

180

 

 

 

 

 

 

 

 

 

 

 

1.0 -
Ph
105
0.8 a
0.6 5
77
0.4 a
0 2 1 122
51 149 154 228
l 1 1 1 27° 30‘
1 (1111 1111 1 1 7 1 1 11 1 1
196
1.0 . 105
0.8 -
0.6 ~ 77
0.4 -
0.2 2 122
51
149164 228
270
1 11 1111]] 11: 1 1 1 111 III I 391
50 100 1 50 200 250 300
m/z

Flgure 2.12.7 N-benzoyl 3'-fluoro-B-phenylalanine methyl ester

82

 

 

1.0 - ‘92

120

0.8 ‘

 

0.6 -

0.4 -

176
162

0.2 - 134 235

65 77
1 I 111102 204217

120

 

 

 

 

 

 

 

1.0]

192

0.8 d

0.6 .

145

176
0.4 '1 91

105
134 162

0.2 -

 

 

 

 

 

 

50 100 150 200 250
m/z

Flgure 2.12.8 N-acetyl 3'-methyl-[3-phenylalanine methyl ester

83

1.0-

0.8 -

0.6 4

0.4 n

0.2 1
51

 

65
mL11111111

 

105

 

168

Ph

 

 

1421212

11L

 

1.0-1

0.8 -

0.5 - 77

0.4 4

0.2 d 51

 

 

1.12.1

 

168

 

 

50

Flgure 2.12.9 N-benzoyl-B-(z-furanyl)-B-alanine methyl ester

84

 

91

 

77

 

 

130

115

103

 

130

115

 

 

103

 

 

.1 111 [(111,

100

 

144
156
1 4
144
156
174
150
m/z

Flgure 2.12.10 N-acetyl- styryl-B-alanine methyl ester

85

 

 

 

 

 

 

186
247
L 11 1
204
186
247
1.1.1— 11 71'
200 250

 

1.0-

0.8 '-

0.6 -

0.4 -

0.2 4

1.0-1

0.8 ~

0.6 a

0.4 -

0.2 ‘

 

162

91 132 146 175 ZTf 219
111.1. .

162

146

202

 

 

234

 

 

 

 

50

Figure 2.12.11 N—acetyl 4'-t-butyI-B-phenylalanine methyl ester.

1 I l

100 150 200
m/z

86

250

 

2.3 CONCLUSION

In conclusion, it is evident that the native PAM is able to accept a broad
array of arylalanines substrates that possess various substituents on the phenyl
rings. However, no definitive trend in the catalytic rate emerged with regards to
strong electron-withdrawing or -donating substituents under steady-state kinetics
conditions. In this scenario substrate release may be, in part, rate-limiting, and
thus may mask other cryptic electron inductive effects associated with the MIO
mechanism. The development of single turnover kinetics analysis of PAM is
currently being investigated in order to further dissect the mechanism.

As the properties of the phenylalanine aminomutase mechanism are
better understood, the strategic integration of amino acid isomerase chemistry
into custom asymmetric synthesis becomes practical. Engineering PAM to
broaden its substrate specificity for the production of novel nonpeptidic B-amino
acids provides building blocks for the design and construction of second-
generation compounds such as taxol and januvia. Predictably, genetic
engineering approaches to modify this biocatalyst can also be directed toward

potentially making PAM more compliant for use in large-scale reactors.

87

 

2.4 MATERIALS AND METHODS
2.4.1 MATERIALS

Styrylalanine and potential allylglycine products were synthesized as
described below. Unless otherwise indicated, E. coli strains and the vectors were
obtained from Invitrogen. The d-amino acid substrate (S)-o-phenylalanine, 2'-
fluoro-(S)-phenylalanine, 3'-fluoro-(S)-phenylalanine, 4'-fluoro—(S)-phenylalanine
and (S)-nor-valine were purchased from Sigma-AIdrich-Fluka. 2'-Methyl-(S)-a-
phenylalanine, 3'-methyI-(S)-o-phenylalanine, 4'-tert-butyl-(S)-q-phenylalanine,
(S)-2-amino-5-phenylpentanoic, (S)-styrylalanine and (S)-allylglycine were
obtained from Peptech (Burlington, MA), 4'-methyl-(S)-q-phenylalanine was
purchased from Advanced ChemTech (Louisville, KY), and 3'-methoxy-(S)-o-
phenylalanine was purchased from Accellant Inc. (Monmouth Junction, NJ). 3-
Amino acid standards (R)-B-phenylalanine ((R)-3-amino-3-phenylpropionic acid),
(Fi)-3-amino-3-(2-methylphenyl)-propionic acid, (Fi)-3-amino-3-(3-
methylphenyl)propionic acid, and (R)-3-amino-3-(4-methylphenyl)propionic acid
were acquired from Peptech, (R)-3-amino-3-(4'-tert-butylphenyl)propionic acid
was acquired from Accellant lnc., (R)-3-aminopentanoic acid and (Fi)-3-amino-5-
phenylpentanoic acid were purchased from BioBlocks (San Diego, CA), (FD-3-
amino-3-(4-fluorophenyl)propionic acid and (Fi)-3-amino-3-(3-
fluorophenyl)propionic acid were purchased from Astatech (Bristol, PA), and (R)-
3-amino-3-(2-fluorophenyl)propionic acid and (R)-3-amino-3-(3-
methoxyphenyl)propionic acid were purchased from Accellant Inc. (S)-2-Amino-

5-phenylpentanoic acid was purchased from Chem-lmpex International Inc.

88

 

(Chicago. IL). These compounds and all other reagents were used without further
purification, unless otherwise noted. Tetrahydrofuran (THF) and dichloromethane
(DCM) were obtained from a dry-still packed with activated alumina that was

pressurized with N2 gas. Silica gel (230-400 Mesh) and aluminum-backed Silica
gel 60 TLC plates, embedded with A254 chromophores, were purchased from

EMD Chemicals Inc. (Gibbstown, NJ.
H-NMR spectra were recorded on a Varian Inova-300 (300.11 MHz), a

Varian VXR-500 or a Varian Unity-500-Plus spectrometer (499.74 MHz) and
were referenced to residual solvent signals at 7.24 ppm for CDCI3). All apparent

coupling constants (J values) are measured at the indicated field strengths.
Coupled gas chromatography/mass Spectrometry analysis was conducted
by loading 1 uL of sample onto an HP 5H8 GC column (0.25-mm inner diameter

x 30 m, 0.25-um film thickness) (Agilent, Palo Alto, CA) mounted on a GC (model

6890N, Agilent) coupled to a mass selective detector (model 5973 inert®, Agilent)

in ion scan mode from 50 - 300 atomic mass units.

89

 

2.4.2. PROCEDURES
2.4.2.1 Heterologous Expression and Purification of PAM

By a cohesive-end PCR method pam cDNA was sub cloned from a
previously described expression vector pET1981His into pET14b in order to
exchange the poly-His epitope from C- to N-terminal-tagging, respectively.
Preliminary purification of small-scale preparations of PAM protein expressed
from pET1981His and from pET14b in E. coli BL21 (DE3) indicated that the N-
terminal-tagged protein bound to Ni-affinity column matrix 8-fold better than the
C-terminaI-tagged protein (<10 % binding). The oligonucleotide primers used for
this sub cloning procedure have been described previously, and the new
expression vector was designated pET14-1981His. This plasmid was used to
transform E. coli BL21 (DE3) cells which were then grown for 16 h in 50 mL of

minimal growth media (1 L contained 12.8 g NaZHPO4-7H20, 3 g KH2P04, 0.5 9
NaCl, 1.0 g NH4CI, 2 mL 1 M MgSO4, 100 (1L 1 M CaCIz, 10 mL 100X BME

Vitamin solution, and 20 mL 20% D-glucose solution) at 37 °C with ampicillin
selection. These bacteria were used to inoculate 6 L of minimal growth media

containing 50 ug/mL ampicillin, and were grown at 37 °C for 5 h to 00600 = 0.6.

Expression was induced by addition of 1 mM of isopropyl-B-D-
thiogalactopyranoside and the cells were grown for 16 h at 18 °C. The remaining
steps were conducted at 4 °C, unless otherwise noted. The cells were harvested
by centrifugation at 5,000 x g (20 min), diluted in 100 mL of resuspension buffer
(50 mM Tris-HCI, pH 8.5), and lysed by brief sonication (five 20-s bursts at 50%

power (Misonix sonicator, Farmingdale, NY)) followed by removal of cellular

90

debris by centrifugation at 15,000 x g (30min). Residual light membrane debris
was removed by centrifugation at 45,000 x g (2 h). The clarified crude
supernatant was partially purified by anion exchange and Ni-affinity
chromatography as described. The fractions containing soluble PAM were
combined, desalted by dialysis, and then used for enzymatic assays. The.
quantity of PAM (~25 ug/mL) was assessed by SDS-PAGE and Coomassie Blue

staining as described elsewhere.

2.4.2.2 Assessing Functional PAM Expression

Natural substrate (S)-01-phenylalanine (500 (M) was added to a 1 mL
aliquot of the protein extract containing partially pure PAM (at ~250 ug/mL) and
incubated for 2 h, and the relative production of (R)-B-phenylalanine was
evaluated. Enzyme preparations yielding ~7.1 pg of derivatized B-amino acid

product per 1 mL assay were used for subsequent kinetic analyses.

2.4.2.3 Screening of productive substrates

Each "unnatural" d-amino acid substrate (Table 2.1), at apparent
saturation (1-2 mM), was preliminarily assayed by in vitro incubation with ~50 pg
PAM overnight. Substrates demonstrating sufficient turnover were then incubated
3 h at 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2 and 3 mM with PAM (~2.50 (19) in duplicate
assays. The enzymaticalIy-derived B-amino acids from the Single stopped-time
incubations were derivatized and quantified as follows: the enzymatically-derived

B-amino acids were derivatized to their N-acetyl methyl esters and each sample

91

was quantified by tandem capillary gas chromatography/electron impact-mass
spectrometry (GC/El-MS). Linear regression analysis was used to convert the
abundance of the base peak fragment ion produced by the product in each
sample by corresponding the abundance of the base peak ion of authentic
sample, derivatized similarly, to the concentration of each standard in a dilution

series from 030 pM, and the data were used to create a concentration

dependent curve, v0 vs [S], in order to separately calculate the KM constants and

V values for each substrate. V values are reported as the ratio of V of the
max rel max
PAM with unnatural substrate to the Vmx with phenylalanine.
The initial rates (v0, nmol/h) were plotted against substrate concentration

[8] on KALEIDAGRAPH (Synergy Software, Reading, PA) to determine kinetic

2
constants (KM and Vmax) of PAM by nonlinear regression analysis (R values

were typically > 0.98). The following amino acids were assayed; allylglycine,
styrylalanine. 2'-fluoro-(S)-phenylalanine, 3'-fluoro-(S)-phenylalanine, 4'-f|uoro-
(3)-phenylalanine and (S)-nor-valine 2'-MethyI-(S)-o-phenylalanine, 3'-methyl-
(S)-a-phenylalanine, 4'-tert-butyl-(S)-q-phenylalanine, (S)-2-amino-5-
phenylpentanoic, (S)-Styry|alanine, (3)-allylglycine 4'-methyI-(S)-d-phenylalanine,
and 3'-methoxy-(S’)-o-phenylalanine. The steric flexibility of the PAM active site

was investigated with 2'-, 3'- and 4'-methyl- and 4'-t-butyl-q-phenylalanines.

92

 

0
COOH COOH
OH
\ COOH COOH
VT'ZOOH \/\er m:

Figure 2.13 Some of the unnatural substrates assayed with PAM.

2.4.2.4 Quantification of Biosynthetica/Iy-derived B-Amino Acids

Processing of the product mixtures after incubation of PAM with the d-
amino acid was analogous to previously described methods. In general the assay
buffer was basified (0.1 M NaOH) until pH z 10, treated with acetic anhydride (or
benzoyl chloride) (2 x 300 11L for 20 min between each addition), and the
reaction was quenched by acidifying to pH 2 with 1 M HCI. The N-acetyl amino
acids were extracted with ethyl acetate (3 x 1 mL), the organic fractions were
combined, and the carboxylic acids were methyl-esterified with diazomethane
treatment. The derivatized amino acid mixture was dissolved in 300 uL ethyl
acetate, and a 1 (1L aliquot of this material was analyzed by tandem gas

chromatography/mass spectrometry (Figure 2.7).

93

2.4.2.5 Determining the Relative Kinetic Constants for Various "Allylglycine"
Substrates

After establishing linearity with respect to a fixed protein concentration and
time, an incubation time of 3 h at 31 °C was chosen for PAM steady-state
kinetics analysis of a homologous series of d-amino acid substrates. Kinetic

constants (KM and Vmax) were determined initially for the natural q-phenylalanine

substrate (at 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2 and 3 mM) in order to later assess

the relative kinetics of PAM (~250 ng) for the surrogate o-amino acid substrates.

2.4.2.6 Synthesis of B-allylglycine
1) 3 egLuiv. CHLCHZMgBr
-78 °C, 1 h

—— + C' 1 equiv. TMSOTL
”@701“ P“ O THF, -78 °C, 1 h
2) 2 M HCI, -78 °C, rt

/ / g/
0 , .
)_N O 0.5 equ|v_ NaOlt/Le, HN O 4.5 equrv. NalO4, rt, 2:11 H2N COOH
Ph — MeOH, 0 °C, 0.5 h —

+

Q

/\
0’ fl COOH

 

 

Phfi—ND’OMe

OTf-

 

 

 

 

Scheme 2.14 The synthesis of B-allylglycine for use as standard of the PAM o-allylglycine
reaction.

2.4.2.6.1 Synthesis of (Ft, S)-1-benzoyI-2-vinyl-2, 3-dihydro-1H—pyridin-4-one

/
o
)—N 0
Ph —
In a 250 mL round bottomed flask was loaded THF (80 mL), stir bar, 4-

methoxypyridine (1.64 g, 0.014 mmol), benzoyl chloride (1.72 mL, 0.013 mmol)
94

 

 

and tris(methyl)triflate (2.64 mL, 0.014 mmol). The mixture was stirred at -78 °C
in an acetone/dry ice bath for 1 h. Vinyl magnesium bromide (30 mL, 0.043
mmol) was then added and stirring continued for another hour at the same
temperature. The reaction was quenched with 2 M HCI and warmed to room
temperature. The formation of product was confirmed by both TLC and 1H NMR.
The product was extracted with ethyl acetate (3 x 50 mL), washed with brine (2 x
25 mL) and dried with sodium sulfate and concentrated in vacuum. The crude
yield was 10.15 g. The product was further purified by flash chromatography by
eluting with a PE/EtOAc solvent mixture. The yield of the yellow (R, S)-1-benzoyl-

2-vinyI-2, 3-dihydro-1-H—pyridin-4-one product was over 90%.

2.4.2.6.2 Synthesis of 2-vinyl-2, 3-dihydro-1H—pyridin-4-one
/

HN O

The (Fi,S)-1-benzoyl-2-vinyI-2,3-dihydro-1H-pyridin-4-one (681 mg, 3.00
mmol) was added to Na (35 mg, 1.56 mmol) dissolved in 9 mL of methanol at 0
°C for 30 min. The reaction was neutralized with 2 N HCI (7.5 mL), dissolved in

EtOAc (25 mL) and extracted with EtOAc (2 x 25 mL). The organic layer was

dried with Na2$O4 and concentrated in vacuo.

95

 

2.4.2.6.3 Synthesis of B-allylglycine
/

H2N COOH

2-vinyl-2, 3-dihydro-1H-pyridin-4-one (5 mg, 0.04 mmol) was reacted with
NaIO4 (38.5 mg, 0.18 mmol) for 2 hours at room temperature. The crude product
was mixed with acidic ion exchange resin (Amberlite IR 120), washed with
distilled H20 until neutral pH and eluted with aqueous NH3 (20 %) and

evaporated to dryness.

2.4.2.7 Synthesis of N-acetyl vinyl-(RS)-fi-alanine methyl ester

/

o
/»\u COOCH3

B-allylglycine (10.52 mg, 0.086 mmol) was dissolved in H20 (5 mL) and

0.1 M NaOH added until pH was ~10. Acetic anhydride (8.97 mg, 0.088 mmol)
was added, and the reaction was stirred for 30 min at 25 °C. The solution was
acidified with dilute 0.1 N HCI and extracted twice with 5 mL ether. The ether
fractions were treated with diazomethane until yellow color persisted, and the

solvent was evaporated to dryness to yield N-acetyl vinyl-(RS)-B-alanine methyl

ester (8.1 mg, 54 % yield, Rf = 0.22 in 90:10 hexane/ethyl acetate, (v/v) on Silica

gel TLC).

96

 

1H-NMR (300 MHz, 00013): 6: 1.69 (s, COCH3, 3H), 2.43 (dd, CH2CHNH 1H),
2.69 (d, COCHZCH, 2H), 3.72 (s, OCH3, 3H), 4.93 (m, CHZCHCH, 1H), 5.19-
5.23 (m, CH=CH2, 2H), 5.92 (ddd, CH2=CH, .1: 18.5, 11.1, 6.0 Hz, 1H).

GC/El-MS, diagnostic ions: m/z 171 ([M+], 2%), 156 ([M-CH3]*, 2%), 140 ([M-
OCH3]+, 6%), 129 ([M-CH2CO]+, 38%), 128 ([M-CH3CO]+, 23%), 112 ([M-

cocha], 18%), 98 ([M-CH2002CH3]+, 14%), 81 [H2C=CHCH=NH2]+.

2.4.2.8 Synthesis of (E)-5-aminopent-3-enoic acid

 

H _ CH2(302H (ilH2304i H _‘ CH2co21-1
H02CH2C H 1“) N3N3 HZNHZC H

 

 

 

Scheme 2.15 Synthesis of (E)-5—aminopent-3-enoic acid.

Trans-B-hyromuconic acid (1.4 g, 0.1 mmol) was suspended in CHCI3 (40

mL) and concentrated H2804 (4.0 mL) added. Sodium azide (0.65 g, 0.01 mmol)
was added in small amounts over 30 min while the mixture was stirred rapidly.
After a further 4 h at 40 9C the CHCI3 layer was decanted from the viscous

residue which was washed again with CHCI3 (30 mL) and the acid layer was

dissolved with water (150 mL) and filtered. The product was derivatized to N-
acetylated methyl ester and assayed on GC/MS. The following peaks were

observed on mass spectrometry fragmentation as m/z, 171

97

 

([H2NCH2CHCHCH2CO2H = M]"), 156 [M-CH3]+, 128 [M-43]+, 140 [M-31]+, 112

[NI-591*, 113 [111-581*, 98 [M-73]*, 99 [M-72]*.

2.4.2.9.1 Synthesis of 4-Bromo-2-pentenoic acid

 

H H
CO H CO H
\H 2 N-bromosuccinimidg W 2
CHCb

H BrH

A mixture of trans-2-pentenoic acid (2 g, 20 mmol) and recrystallized N-
bromosuccinimide (3.56 g, 20 mmol) in carbon tetrachloride (40 mL) was
magnetically stirred and refluxed until the suspension rose to the surface (24 h).
The mixture was then cooled on ice, filtered and evaporated to oil. The product

was purified by crystallization from 60-80 0C light petroleum/cyclohexane to give
4-bromo-2-pentenoic acid in 60 % yield (lit). 1H-NMR (300 MHz, CDCI3): 6 = 7.24
(dd, CH=CH, J = 13, 6.5 Hz, 1H), 6.00 (dd, CH=CHCH, J = 13, 0.7 Hz, 1H), 4.75

(multiplet, CHCHCH3, J = 7 Hz, 1H) and 1.86 (d, CHCH3, J = 5.5, 3H).

2.4.2.9.2 Synthesis of (E)-4-amino-2-pentenoic acid

H H
CO H CO H
W 2 NHQ _ W 2
H THF, 12 h H

Br NH2

4-bromo—2-pentenoic acid (3.56 g, 20 mmol) was dissolved in

Tetrahydrofuran (THF, 15 mL) and added dropwise to liquid ammonia (200 mL)

98

over 3 min with vigorous stirring for 12 h. The solvent was evaporated and the
crude product was dissolved in water (20 mL), acidified with 1 M HCI and

extracted with diethyl ether (2 x 20 mL). The aqueous layer was adsorbed on a
Dowex 5W (H+) column (40 mL). The column was eluted with water until neutral
and then the amino acid was removed with 1 M ammonium hydroxide. The

ammonia wash was evaporated to dryness and the product was purified by

crystallization from ethanol to give trans-4-amino-2-pentenoic acid in 80 % yield.

Mp = 1987 90. 1H-NMR (300 MHz, D20): 6 = 1.42 (d, CH30H, J = 16, 3H), 4.10

(CH3CHCH, J = 10, 1H), 6.05 (d, CH=CHCO2H, J = 16, 1H), 6.60 (dd,

CHCH=CH, 1H).

2.4.2.10 Invivo Screening of Amino acids

Phenylalanine analogues were fed to a 50 mL culture of transformed BL21

(DE3) over expressing the PAM enzyme. The culture was initially grown to ODaoo

of 0.7 at 37 0C with ampicillin selection and expression was induced with 1 mM
isopropyl-B-D-thiogalactoside (IPTG). Incubation was continued for a further 18
h. The substrate concentrations ranged from 1 - 5 mM. The assay for the
formation of the B-product from the in vivo biosynthesis was carried out by

centrifuging at 5,000 x g for 20 min. The supernatant and cell pellet were treated

separately.

99

 

2.4.2.10.1 Treatment of the cell pellet.

The cells were resuspended in 2 mL of phosphate buffer and lysed by
sonication (5, 20 s bursts at 50% power [Misonix sonicator, Farmingdale, NY]).
The cell debris was pelleted by centrifuging at 5 000 x g for 20 min. The
supernatant containing the amino acid residues were basified to pH above 10
with 1 M NaOH, followed by addition of 3 equivalence acetic anhydride. After 30
min the solution was acidified using 1 M HCI and the N-acetylated product was
extracted with ethyl acetate (3 x 15 mL). The solvent was evaporated to dryness
and the solid residue was re-dissolved in a minimum volume of EtOAc, cooled in
dry ice and treated with diazomethane to a faint yellow coloration. The

derivatized amino acids were analyzed by GC/MS.

2.4.2.102 Treatment of supernatant
The supernatant was basified with 6 M NaOH and derivatized as

described above.

2.4.2.11 Investigating the racemase in E. coli
In order to investigate the action of the racemase in E. coli a D-[ring, 2H5]

phenylalanine was incubated with E. coli BL21(DE3) transformed with the pam
plasmid as described above. Further effect of the D-isomer on the PAM reaction
was carried out by incubating various concentration of this amino acid with pure

PAM.

100

 

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114

CHAPTER 3

Characterization of Phenylalanlne Aminomutase (PAM): Conflrmlng the
Presence and Role of 3, 5-Dihydro-5-Methylidene-4-H-Imldazol-4-one (MIO).

3.1 INTRODUCTION
The 3,5-dihydro-5-methylidene-4H-imidazol-4-one(MIO) prosthetic motif
present in Phenylalanine ammonia lyase (PAL) and histidine ammonia lyase

(HAL) acts as a Lewis acid or electron sink during catalysis."18 The MIO is auto

catalytically formed from the triad sequence of alanine, serine and glycine (ASG)

which is also present in enzymes like PAL, tyrosine aminomutase (TAM) and

HAL.19'28 Earlier studies had proposed a dehydroalanine formed serine

29-31

dehydration as the electrophile. X-ray analysis of the HAL from

31 -37

Pseudomonas putida showed the true electrophile to be the MIO. The phenyl

ring or the amine group of the substrate is proposed to act as the nucleophile.
The long known mechanism of PALs suggested the amino group of the o-amino
acid is the nucleophile. However some modeling calculations and broad
substrate specificity studies (see Chapter 2 for more details) by Rétey and co-

38-44

workers point to the aromatic ring of the substrate acting as the electron

donor ( discussed in detail in Chapter 5).

115

 

 

HO k, 0H :Base
W Hf-
NH6 0 “‘ O
K‘ ,, /N H /N H

Scheme 3.1 Shows the formation 3, 5-dihydro-methylidene-4H-imidazol-4-one prosthetic group
(far right) which occurs in the active site of HAL from P. putida. The alanine-serine—glycine triad
undergoes cyclization and dehydration to form the MIO motif.

Christenson and Calabrese working on different enzymes (TAM and HAL,

respectively) were able to confirm that the MIO binds to the amine group.2’14

Amid all the controversy what is universally acceptable is that the MIO promote

the elimination of NH3 and H+ from arylalanines substrate by lowering the pKa of

the B-hydrogens of the amino acid. The pKa of the B-hydrogens drop by 30 units
upon MIO binding, 26 of those units are attributed to the six positive helices
pointing towards the active site and the other 4 units are due to the contribution

6,31

of the MIO. That observation minimizes the role of the MIO because it implies

that in its absence the reaction still goes on to completion. However studies in
which L-5'-nitrohistidine is used as a substrate for HAL from Pseudomonas
putida react with MIO-less mutants (in which the serine of the ASG triad has
been replaced with glycine or threonine) and with wild-type HAL to the same
extent.45 Similar observations have been made with PAL from R. tolouride in
which serine-202 was mutated to alanine, glycine and threonine by site directed

mutagenesis. These results may not favor the mechanism by which the amine is

116

the nucleophile and prefers the one involving a transient Friedel-Crafts-like
alkylation via aromatic ring. The nitro- group is believed to act in place of the MIO
by its electron withdrawing effects which makes the B- protons acidic enough to
be de-protonated by a general base (histidine, cysteine or tyrosine) to set an
E1cb elimination of ammonia (Figure 3.5). The electron withdrawing nature of the
nitro group would still result in acidic 6 hydrogen even if the amine was the

nucleophile as opposed to the phenyl ring.

more acidic
thaninthe
unbound \H O

substrate ;
or”:
(’3 biz/E -H-Enz
NYN\/

Figure 3.2 shows the amine group of L-a—phenylalanine bound to the MIO prosthetic group. The
pH of the B-hydrogen drops by nearly 40 pKa units.

In this work the role of the MIO on the PAM reaction is investigated by
mutating serine-176 to alanine, threonine, glycine and cysteine, comparative
kinetic assays between wild-type and mutant enzyme, with various q-amino acids

will be employed.

117

Characterization and optimization of overexpression of PAM
The full-length pam cDNA has open reading frame of 2094 bp, which

encodes 698 amino acid residues. It has a calculated molecular mass of 76,530

kDa and an experimental mass of 80 kDa by SDS-PAGE analysis.18 The
turnover kcat has been reported as 0.015 s'1 with KM value of 45 pM measured

with the natural substrates. Inhibition studies conducted mainly on phenylalanine
ammonia lyase (PAL), histidine ammonia lyase (HAL) and tyrosine ammonia
lyase (TAM) have demonstrated the presence of the catalytic moiety

46-48

(methylidene imidazolone). The functionality of the exocyclic double bond of

the MIO makes it reactive with nucleophiles. The requirement for a electrophile in
ammonia lyases was first reported in 1967 when these enzymes were inhibited

by several nucleophilic reagents, such as hydroxylamine, hydrazine,

bromocyanide, sodium borohydride and cysteine.”51 The inhibition of both PAL

and HAL with cysteine was determined at a pH of 10.5 in a N32003/HCO3 buffer.

The reaction is reversible under anaerobic conditions and irreversible in
presence of oxygen. The cysteine-MIO complex (Scheme 3.1) absorbs
maximally at a wavelength of 340 nm and which has become a qualitative test for

the presence of the MIO.

118

l

 

H H
> 23
- NH
8 2
NH2 H0 H
HO H O
L-Cys/OZ
pH 10.5

 

v 3: f0
0
3:0 Disulfide bond / N
formation N r\_
N I N /
/ c N ___ ) 0
N O —
f" '

HNH

H l
HN HOOC HOOC>—/

HOOC"; s 3 S”
s
HZNSfiTCOOH

Scheme 3.3 The reaction of cysteine with the MIO moiety of PAL or HAL at pH 10.5. The
reaction is irreversible in the absence of oxygen and is stable under aerobic conditions 49.

Walker and coworkers have demonstrated that PAM from Taxus
canadensis is inhibited by both cyanide and sodium borohydride,18 but no studies

have been reported on whether cysteine has an effect. Further investigations will
be conducted to confirm the presence of the MIO in PAM by utilizing cysteine as
an inhibitor and measuring the formation of the cys-MIO complex by UV/visible

spectroscopy.

119

S52. 53

The active site of PAL from R. toruloide show that phenylalanine is

anchored in the active site by arginine and lysine on the carboxylate end of the
molecule, the phenyl ring forms hydrophobic interactions with leucine and
isoleucine residues, the amine group interacts with a tyrosine and histidine. The
amine group of the substrate must be neutral for the conversion to occur,
suggesting slightly basic conditions in the active site. The amine is striped of a
proton by a basic residue of the enzyme prior to entry into the active site. Both
suggested mechanisms (amine or phenyl ring as nucleophile) for PALs and HALs
point to a requirement of a base which de-protonate the 8 hydrogen to set an
E1cb mechanism which results in the elimination of ammonia.

Previous studies on PAM have proved that although the primary reaction
is the conversion of 2-(S)-a-phenylalanine to 3-(R)-B-phenylalanine the reverse

reaction in which 3-(R) and 3-(S) act as substrates produce only 2-(S)-

18

phenylalanine. We intend to carry out an investigation to confirm the

reversibility of the PAM reaction in order to determine the equilibrium constant
and chirality of the reaction products.
Although PAM T. canadensis has a 99 % amino acid sequence identity

with the functionality similar PAM T. chinensis the later has a relatively high KM
value of nearly 1 mM.54 This large discrepancy could be due to the different

assay conditions and detection parameters used to analyze the properties of
PAM in the separate studies. The studies on PAM T. chinensis were carried at
concentration of 5 mM to 35 mM using HPLC and LCMS techniques. Walker and

coworkers used concentration in the micromolar range but utilized GCMS which

120

is a more sensitive detection method. The GCMS method sensitivity is due to its
low detection limits being in the nanomolar range, and is useful in identifying
analytes based on the m/z fragmentation patterns. However, the technique
requires that analytes be relatively volatile, therefore amino acids are derivatized
to the N-acetylated methyl ester forms using acetic anhydride diazomethane (as
shown in scheme 3.3). The multiple steps involved could potentially introduce
both systematic and random errors in the results. Beside derivatizing analytes for
GCMS analysis; another complimentary technique is to use a chiral column on

HPLC. No expensive sample preparation is required and sensitivity and
selectivity can be enhanced by derivatizing with O-phethaldehyde (OPA).55'68 The

HPLC method which excludes the derivatization steps will be a faster and less
costly technique than GCMS providing a better linear dynamic range of up to 35

mM.

121

 

0 69
D’. D 8) H3N,‘ D O ‘ H2N, D O
NH PAIL/1 8) NaOH (1 N) t (89
G) 3 31 C D PH>10 D
(CH3CO)20

o o
HN,.DO + Y 0 Y

 

HN o HN o 0
OCH3 EELS. ’- $1- H+ (pH < 2) I, 5)
o o OCHB 2. Extract with EtOAc
o
180 3. CH2N2

1+

o
HN’U\

D
149

b c-l

 

 

Scheme 3.4 The PAM reaction is assayed by incubating the enzyme with the substrate at 31 °C.
The reaction is quenched with 1 N NaOH before adding acetic anhydride followed by
diazomethane after extraction with ethylacetate at pH 2.

The acquisition of pure enzyme is a determinant factor in enzymology,
especially for structural and kinetics studies. The enzyme is overexpressed in E.
coli as host and requires purification to over 95% homogeneity. There are
essentially four factors that determine the extent of protein expression: the host
in most cases E. coli or yeast; the temperature of induction; the carbon source;
and the induction procedure. The expression of PAM at 18 °C or higher in E. coli
cell line BL21 (DE3) LysS has previously resulted in low yield of PAM (less than
10% of the whole cell protein content). Here we report how the effect of the

combination of low temperature and cell line with a built in rare codon (arginine,

122

proline, isoleucine and lysine) optimization mechanism increased PAM

expression.

I Investigating the BASIC residue required during phenylalanine aminomutase
catalysis

The major role of the prosthetic group methylidene imidazolone (MIO) is

believed to be reduction of the pKa of the B-hydrogens of the substrate, for easy

abstraction by a general base.36 Enzymes which exhibit similar behavior are

phenylalanine, histidine and tyrosine ammonia lyases (PAL, HAL and TAL).69'75

TYF363 in PAL 3 toruloides,53 Tyr300 in TAL from R. sphaeroides 2' 12' 76

equivalent to Try322 in PAM Taxus. Canadensis 77 based on sequence
alignment (Figure 1.4). Mutations of the Y280, 300 and 363 to phenylalanine

showed no or reduced activity”. In PAL the tyrosine promotes the breakdown of

the MlO-NH2 complex by donating a proton to the -NH2- group (Figure 3.6).

Try363 is near a positive helix which increases its acidity and its role as a general
acid. Its resting state is in the neutral form with a pKa~ 10. The phenolate form of

tyr363 is stabilized by the nearby guanidinium group of Arg366 which also

interacts with one of the positive helices. Dissociation of the MlO-NH2 complex

and release of NH3 and Cinnamic acid to the bulk solvent put the enzyme in its

resting state. The general base will be most essential if the amine is the
nucleophile. If the phenyl ring was the nucleophile there won’t be any need for a

base because the stabilization of the ipso positive charge will not necessarily

123

require the loss of the B proton through abstraction by an enzymatic base. Any
water in the active site will be able to act as a general base, to remove the highly

acidic proton.

H’ ‘3 '
0'
03 H

H H HWOH H H
\‘ - ‘,. .
020‘ "WOH= 620“/- waft/C}—OH

HZPV H H211 ”\{HZ
MjO M10 M10

ll

Flgure 3.5 The mechanism of SgTAM based on established crystal structure. The tyrosine
substrate is shown bound to the MIO via the amine group. Tyrosine 63 acts as a general base to
de-protonate the pro-(R)-B-hydrogen stereospecifically. A coumarate is formed as an
intermediate. The MIO shuttles the amine group to the [3 position, the subsequent breakdown of
the MIO-amine complex results in release of the B-amino acid as a product.

124

Hi896 lle 431

r
(O
C
A
I
I
I
I
I
I
I
I I
I
I! ’I I”
I l’
I I I
I ’ l I
I I I’ I’
' ’ I I
I I
I I I
I ’I
I”
I,’
’I
’I
I
O/ \ .
' \
I \
I
I
I
I
I
I
I
I
I
I
:‘z
:3
$
A
(O

k """"""" 0
Val 248
, , N / radé/ ’ NC:— Ar9325
Leu225 /_L/: {L

HN

/ £0 Phe371

Asn219

Figure 3.6 The active sites of PAL R. tolouride at the top showing the binding of the substrate at
the active site.

In this work, we intend to demonstrate that tyrosine 322 from PAM act as
a general base in the mechanism, by studying the activity of various mutants of
PAM; Y322H, Y322F and Y322A. The last 2 mutants are expected to be inactive
towards the natural substrate if tyrosine 322 of PAM is essential during the
catalysis. If there is no difference in activity, it may suggest that the tyrosine 322
is not very essential and the mechanism could be involving a short lived
carbocationic transition state, which is neutralized by the spontaneous loss of the
B-protons. Although histidine may be a better base than tyrosine, the shorter
bond length may not allow for the shuttling of the proton which is perceived to
take place during the course of the reaction. The contribution of the cysteine 107

in PAM will also be studied. Two mutants will be constructed for that purpose,

125

0107A and C107H. Mutant C107A is expected to show decreased activity and

C107H showed have higher if the cysteine is considered to be acting as a base.

126

3.2 RESULTS
3.2.1 Optimization of the over expression of phenylalanine aminomutase.

The recombinant plasmid of the pam gene and pET 14b was used to
transform Eco/i cells line BL21-CodonPlus-RIPL competent with rare codon
optimization for arginine, isoleucine, proline and lysine. The expression of PAM in
either BL21(DE3) or BL21-CodonPlus-RIPL cells at 18 °C resulted in low yields
(Figure 3.7). The expression temperature was reduced to 16 °C with IPTG
induction (Figure 3.8). The recombinant protein was purified using a Ni-NTA
column. The elutions collected from the column were analyzed by SDS-PAGE

and Coomassie blue staining (Figure 3.9).

75 kDa —>

123456789

Figure 3.7; SDS-PAGE of proteins expressed in BL21-CodonPlus-RIPL cells transformed with a

pam plasmid at 18 °C or 37 °C. The PAM protein with a size of 76 kDa is barely distinct from the
rest of the cellular proteins pointing to weak expression. Lane 1 the Lonza protein marker with the
75 kDa bark shown on the left. Lanes 2-9 are whole cells from different flasks and treated by
spinning the cell cultures at 13 000 rpm, boiling the pellet in BME for 20 min.

127

q
' i. , .'."'““y I
-1 I

.g

. .. ‘ ’
- . - . t , ‘
.' it ' A.‘ I 't!
. o- - .T ,‘
.‘JS' 5‘... . ,1 _.
‘fi ~ ‘l ‘2 5' WW

3 4 5 ~€57 8 91."

r it...

 

l
.’ I
(If. . ,
e. ~“ ““4.
. .. '.'..

mt??? «we vet-ex

   

Flgure 3.8; SDS-PAGE of proteins expressed in BL21-CodonPlus-RIPL cells transformed with a

parn plasmid at 16 °C. The PAM protein with a size of 76 kDa (lanes 1,3,4,5 and 6) was over
60% of the cellular proteins suggesting a successful over expression. Lane 8 is the protein ladder
(Lonza) and the 75 kDa marker is indicated on the right.

75 kDa —> : _ ..

Flgure 3.9 The SDS-PAGE gel of elutions of PAM from the Ni-NTA column. The PAM has a
measured mass of about 75 :I: 5 kDa based on the protein ladder in lane 1. The protein was over

expressed in BL21-CodonPlus-RIPL at 16 °C and induced with 1 mM IPTG.

3.2.2 Enzymatic Assay Techniques

PAM was confirmed as activity if 10 ug of the enzyme converted 1 mM of

the natural or unnatural amino acids such as 4'-NOz, 4’-F, and 4'-MeO, -

phenylalanine as substrates to 52% of product overnight. The activity of wild-type
PAM with electron-withdrawing and donating substrates was assayed by

incubating 50 pg of enzyme with 1 mM of substrate at 31 °C for 3 h. All the

128

substrates tested were reactive as expected, the flouro-substrate was the most
reactive with a VMAX of 350 units/h/mg and the nitro- was the least reactive at
150 units/h/mg which even less than the natural substrate at about 200 units.
The higher reactivity of the flouro-substrate has been reported previously,78

although its relative rate compared to the natural product was much higher (95

times faster) than what we report here (2 times faster).

400

350 - + +

300 1

 

250 ~

200‘ E ‘

150 ~

Vmax (units/h/mg PAM)

100 ~

50~

 

 

 

 

 

 

 

 

 

 

 

4MeO 4F D8 4N02

Figure 3.10 An activity assay PAM (50 ug) incubated for 3 hours. The 4'-methoxyphenylalanine
and 4'-Flourophenylalanine is converted to over 50%, the universally deuterated phenylalanine
and the 4’-nitrophenylalanine are at 30% conversion.

3.2.3 The PAM pH dependence

I The pH dependence of PAM was determined by measuring the activity at
different pH values. The pH of PAM was adjusted from 8.5 to 11 and the
substrate added and incubated at 30 °C for 1 h. The samples were derivatized as

129

before and injected into the GCMS. The pH profile (Figure 3.11) shows a rapid
decrease in PAM activity as pH decreases and a more tolerance of PAM to more

alkaline conditions. PAM activity peaks at a pH of 8.5.

 

50
45 4
40 ~
35 .
30 ~
25 ‘
20 -
15 ~
10 ~

% mol B-phe

 

 

 

6.5 7 7.5 8 8.5 pH 9 9.5 10 10.5

Flgure 3.11 The pH dependence of the PAM reaction. The activity increases rapidly form pH 6.5
and peaks at pH 8.5 before it gradually decreases at higher pH.

3.2.4 Cysteine Inhibition studies

The reaction of cysteine with PAM was monitored spectroscopically and
by activity assay, measuring the amount of B-product formed during the reaction.
Samples were collected every 10 min and derivatized as described elsewhere

and analyzed by GCMS.

130

3.2.4.1 Spectral evaluation

The reaction was monitored by scanning from a wavelength of 300 nm to
400 nm at a rate of 1200 nm/mn. The spectrum show a peak at about 340 nm
(Figure 2.12) which is typical of most enzymes carrying the autocatalytically

synthesized methylidene imidazole cofactor.

 

3.1
3.0
2.9
2.8
2.7

2.6

 

Absorbance

2.5

2.4

 

 

2.3

 

300 305 310 315 320 325 330 335 340 345 350 355 360 365 370 375 380

Wavelength (nm)

Flgure 3.12 Spectra of the formation of the MIO-cysteine complex monitored over a period of 60
min. The spectra were at 10 min intervals after incubating with 10 mM L-cysteine and show
increase of absorbance at 340 nm with time.

3.2.4.2 Activity Assay with cysteine inhibition
The activity of the cysteine inhibited PAM was monitored for 60 min with
samples at 10 minutes intervals. A sharp decrease in activity is observed falling

to less than 5% (black line in figure 3.13). The activity was restored to 100% after

dialysis, to remove cysteine.

131

 

 

 

 

 

 

 

 

 

120
e “’0‘ + +
.2
2 801
2
<
a. so -
Q)
.2
‘63
a 40-
o:
0
°\ 20 - H
o 1 I V I 7 I j I
1 o 10 20 30 4o 50 60
Time (min)

Figure 3.13 The effect of cysteine inhibition on PAM activity. PAM was reacted with 10 mM

cysteine at 30 °C for 50 min. After 50 min the cysteine was removed by filtering through a 30 kDa
cut of Amicon filter and the activity of PAM was fully restored as measured by the relative activity
of the free versus the uninhibited enzyme.

3.2.5.1 Expression and Purification of Recombinant Wild-type PAM and Mutants
8 176A, S1760, 81766 and S 176T.

The mutants S176A/GIG and T were over expressed in
BL21(DE3)CodonPlus-RIPL cells and purified using Ni-NTA technique to over
95% homogeneity. The elutions from the NTA column were denatured by heating
at 95 °C for 15 min in 50% SDS-BME solution. About 0.1-01 pg of protein was
loaded on polyacrylamide gel (20 uL well) prepared by mixing; water (3.4 mL),
1.5 mM Tris-HCI (2.5 mL), 20% SDS buffer (50 uL), 30%/0.8% bis/tris-

acrylamide (4 mL ), 10 % ammonium sulfate (10 uL) and TEMED (5 uL). The

132

electrophoresis was run in SDS buffer at 180 V for 2 hours; the gel was stained

in Coomassie for 1 h and de-stained in water/methanol/acetic acid (Figure 3.15).

75 kDa

1234567891011

Figure 3.14 The SDS/PAGE gel with Coomassie staining showing over expressed wild-type PAM
(lanes 2 and 3), S176A (lanes 4 and 7), S176G (lanes 5 and 8), S176T (lanes 6 and 9), showing
that the 4 proteins are of the same size (75 1 5 kDa).

3.2.5.2 Difference Spectra between wild-type PAM and MIO-less mutants

The difference spectra in which the mutant S176A, G and T were used as
blank to measure the UV-visible spectrum of wild-type PAM is shown in figure
3.15 with a absorption maxima at about 308 nm which is characteristic of
phenylalanine ammonia lyase enzymes which have an MIO moiety. The
absorption at 308 nm increases with increase in concentration of the wild-type

PAM.

133

 

0.3
0.2
0.1
0
-O.1
-0.2

-0.4
-0.5
-0.6
-0.7

 

-o.3 \

 

 

 

——0.2 mg/mL
—0.4 mg/mL .
0.8 mg/mL

~ ~2.0 mg/mL

 

 

 

 

260

280

I I

300 320

wavelength, nm

1 I

340 360

 

Figure 3.15 The difference spectra between the mutants S176A/GIT versus wild-type PAM
exhibiting a Amax at 310 nm which increase in strength with increase in the wild-type enzyme

concentration.

3.2.5.3 Activity Assay for the mutant 81760

The mutant S176C (50 pg) was incubated with 1 mM mixture of L-
phenylalanine, 4'-MeO-, 4'-NOz-, 4'-F-phenylalanines for 3 h. The assay mixture

was derivatized as described above and the rates were measured as mol %

conversion of the substrate to the product.

134

 

100

 

90 l
8, - +
70 1
60 1
50 -
4° ‘ ~+~
30 ~
20 -

10 4
4MeO 4N02 4F 4H

Vmax (units/h/mg PAM)

 

 

 

 

 

 

 

Figure 3.16 The activity of the mutant 81760 was assayed by incubating with various amino

acids for a period of 3 hr. The natural substrate showed more reactivity than the 4'-F-Phe which
exhibit higher with wild type PAM.

3.2.5.4 The Linewevear-Burk plot for wild-type PAM vs 4'-nitrophenylalanine

In order to calculate the accurate values for the Vmax and KM for the PAM-

4'-nitrophenylalanine reaction, a Lineweaver-Burk was plotted (1N vs 1/[S]) and

the values were 0.4 unit/h/mg and 286 pM respectively (Figure 3.17).

135

  

 

 

g 1 y=1.173x+3.989
. 2 _
at R .0987
‘TA 7 1
‘2” 6 .
§ 5 1
O
E
..\° 3 ‘
E 2*
v- 1 _
-4 -2 O 1/[S],M" 4 6

Figure 3.17 The Lineweaver-Burk plot for wild-type PAM vs 4'-nitrophenylalanine. The calculated
KM value is 0.3 mM.

Table 3.1 Kinetics parameters of wild-type PAM with substrates L-phenylalanine, 4'-NOz-L-
phenylalanine, 4'-F-L-phenylalanine and 4'- MeO-L-phenylalanine.

 

 

 

 

 

Substrate Vmax, units/mg KM, mM
Phenylalanine 200 :1: 18 009710.008
4'-NOg-L-phenylalanine 166 :l: 18 0.286 :I: 0.058
4'-F-L-phenylalanine 360 :l: 30 0.075 :1: 0.007
4’-MeO-L-phenylalanine 346 :1: 24 0.41 :1: 0.008

 

 

 

 

 

3.2.5.5 Kinetics for mutant S176A with 4'-NOz-L-Phe as substrate

To assess the reaction between the mutant S176A with the a-
nitrophenylalanine as substrate, 50 pg of the enzyme was incubated with varying

ConCentrations of substrate (ranging from 100 pM to 25 mM) for 2 hours (based
136

on time course assay). The Lineweaver-Burk plot for the mutant S176A versus

the 4'-nitrophenylalanine is shown in figure 3.19. The calculated KM value was 1

 

 

mM.
60 -
y = 4.476x + 0.616
1 5° ‘ R2=0.989
3’ 4o -
E
3 30 1
o\°
v 2 —(
5 o
10 -
/
oz 0 0.2 0.4 0.6 0.8 1 1.2
1/[S] (mM'1)

Figure 3.18 Plot of 1/Vmax versus 1/[8] for the reaction between the mutant S176A and d-4'-
nitrophenylalanine as substrate. The KM is 1 mM.

3.2.5.6 Activity Assay of mutant 81766

The mutant S176G was reactive with electron-withdrawing substrate such
as 4'-nitro- and 4'-flouro-phenylalanine but was not reactive with neutral or
electron donating substrate such as phenylalanine and 4'-methoxy-
phenylalanine. The nitro substrate was relatively 5 times more reactive than the
flouro substrate suggesting an increased electron-withdrawing with the former

compound (Figure 3.19).

137

 

4N02

%mol clnnamate

4H 4MeO

 

 

   

 

Flgure 3.19 Activity of mutant S176G as measured by the amount mol% of cinnamate formed
from the o-amino acids (4'-nitrophenylalanine and 4'-flourophenylalanine). The B—product was
below the detection limit. The results suggest the influence of the electron withdrawing groups on
the phenyl ring of the substrate.

3.2.5.7 The Michael-Menten plot for mutant 81760 with the 4'-nitrophenylalanine
substrate.

The Vmax and KM for the mutPAM 81760 with the 4'-nitrophenylalanine

substrate were determined using the Michael-Menten plot of 1/V versus 1/[S]

(Figure 3.21). The Vmax values was calculated as 12.5 units/h/mg of the mutant

and the KM was 0.91 mM.

138

 

90*
804
701
601
$01
404
301
20*.
101%

 

V (units/h/mg mutS176C)

 

049
0

12

[4N02] (mM)

 

 

Flgure 3.20 The Michael-Menten plot for mutant 81760 with the 4'-nitrophenylalanine substrate.

3.2.5.8 Comparison of the kinetic parameters of wild-type PAM against the
mutants S176A/C/G and T with both the natural and 4'-nitrophenylalanine
substrates.

Except for the S176T mutant all other mutants were reactive with 4'-

nitrophenylalanine as substrate Table 3.2).

Table 3.2 Comparison of kinetic parameters from wild-type PAM, the mutants S1766, S176A,
8176T and 81760 with 4'-nitro henylalanine as substrate.

 

 

 

 

 

 

 

 

K

, 22:22“
(mM)

thAM 0.9110065 32512.5 2712.2 0.6310072

8176A 1.01009 - 013510.013 -

£1760 1.110.01 4.221004 0.5110045 01210.01

 

 

139

 

3.2.5.9 Investigating the base involved in PAM catalysis

The assay to check the activity of the of the five mutants Y322A, F, H and
C107A, H was carried out by incubating the enzymes with 1 mM of the natural
substrate. Only mutants C107 A and C107F showed activity (Figure 3.22),
suggesting that Y322 is essential during PAM catalysis. He Y322F could

potentially be used to study the mechanism of PAM.

—L
U" N
1 L

%mol/h/mg

P
01
1

 

 

O

4H 4F 4H 4F 4H 4F

4F 41" 4F

| C1 07A C1 07H Y322A Y322F Y322H

4H

 

Figure 3.21 The assay to check the activity of the of the five mutants Y322A, F, H and C107A, H
was carried out by incubating the enzymes with 1 mM of the natural substrate (4H) and 4'-flouro-
o-phenylalanine (4F). Only mutants C107 A and CI 07F showed activity.

140

3.3 DISCUSION

The over expression of phenylalanine aminomutase (PAM) a plant
enzyme as a recombinant protein in E. coli presents a special challenge because
of some codons which are unique to plants but rare are in bacterial cells. The
codons for arginine, isoleucine, proline and leucine are conspicuously absent in
E.coli and results in low expression of heterologous proteins. There are two
approaches in overcoming the codon utilization dilemma that is; 1) rare codon
optimization through targeted mutation of the gene by substituting the plant
codons with E. coli compatible codons, 2) utilize E. coli cells with a plasmid
carrying the plant codons. The two approaches have been applied in our lab with
a measure of success. However the disadvantage of the gene optimization
technique is the cost. The expression of PAM in BL21-CodonPIus-RIPL cells
resulted in the sixfold increase in the ratio of the recombinant protein to the non-
recombinant cellular proteins (Figures 3.8 versus 3.9). A low expression of the
recombinant protein makes the purification steps very difficult and time
consuming and may present interferences to the assay reaction. This is particular
noticeable for the PAM reaction because the use of impure enzyme results in the
epimerization of the substrate because of the presence of a phenylalanine
racemase from E. coli. When crude PAM enzyme is fed with an octa deuterated

substrate both the unreacted 0 phenylalanine and the corresponding [3 product
show loss of a 2H which is not observed when the same reaction is carried out

with the pure enzyme. With pure enzyme the diagnostic peak for the [3 product is

186 m/z) but with impure enzyme a 185 or 184 peak is observed.

141

79-86

The mechanism of phenylalanine racemase involves the loss of an 0-

hydrogen of the L-phenylalanine to form D-phenylalanine (Scheme 3.22)

(8
H3

0 o
0 ATP WED-AMP W,02AMP W011
OH = u = = E
NHz H NHZ
NH2 ' a
\

”2'?! H9
H2 \ D-Phenylalanine

Scheme 3.22; A summary of the epimerization reaction catalyzed by the phenylalanine
racemase, the imine intermediate attacks a proton from both faces to form a racemic mixture of L
and D phenylalanine.

L-Phenylalanine

The combination of low temperature and use of “CodonPlus-RIPL” cells
provides the best conditions for PAM overexpression. The optimum temperature
for PAM expression is 16 °C. The low temperature slows down the metabolic
pathways of E. coli allowing only the heterologously protein to be expressed
preferentially since it is encoded by a different plasmid from the bacterial
chromosome. Lowering the temperature below 15 °C up to 10 °C may still
produce better results but is likely to be counterproductive because it lengthens
the incubation time and making the whole process of protein purification
unnecessarily long.

The equilibrium of the PAM reaction is 1.1 at 31 °C in favor of product
based on assays with the natural substrate. The Jansen group reported an
equilibrium of 51/4987 which is the same as what we reported. Chen and
coworkers reported an equilibrium value of nearly 50% for the tyrosine
aminomutase reaction which is mechanistically similar 0 PAM.54 The relatively

low conversion of L-o-amino acids to corresponding B-product in plants and

142

bacterial may be a natural selection process to prevent a competing reaction in
which the formation of trans-Cinnamic acid a precursor of many structural and
metabolites of both plants and animals will not be compromised.”99 However the
equilibrium mixture produced presents challenges in the separation and
purification of the amino acids at the end of the reaction given the close physical
and chemical properties of d and B aromatic amino acids. However the
separation can easily be accomplished using HPLC (Figure 3.24). D and L
phenylalanine racemic mixtures can be separated with a high resolution by
utilizing the chiral column. The B- and a- are less chemically equivalent and
should be separable even with a higher resolution than the D and L isomers.
With the capability of high injection volumes and high concentrations of the
amino acids HPLC technique can be use in preparative methodologies to give

99% enantiomeric excess.

143

 

500

L-o-phenylalanine
400 -

300 ‘

D-a-phenylalanine

200 ~

”Z. 1L0

mAU

 

 

 

 

 

 

 

.0
O
0

TIME, min

Figure 3.23 The HPLC spectrum of a mixture of D and L phenylalanine. The amino acids were
separated with 100% methanol isocratic gradient and no derivatization was necessary.

The identification and confirmation of the presence of the MIO in PAM is
central in determining its mechanistic pathway. PAM shares a peptide sequence
homology with PAL and HAL which have been demonstrated to utilize the MIO
mechanistically. The simplest and most direct way of verifying the presence of
the MIO is the reaction with nucleophiles such as the cysteine inhibition reaction.
As shown in Figure 3.13 PAM forms a complex with cysteine which absorbs at
338 nm typical of MIO carrying enzymes. Similar inhibition studies carried out

with sodium borotetrahydride or bromocyanide confirms the complete inhibition of

144

PAM.100 The fairly high pH conditions required for this reaction suggest the need
for a free thio group which nucleophilically attacks the MIO in the enzyme active
site. Independent pH studies of PAM shows a tolerance of basic conditions which
could point to the requirement of a free amine of the substrate as the nucleophile.
The activity of PAM drops sharply at low pH which could be due to; formation of
an ammonium form of the substrate making it difficult to bind into the activity site;
inhibition of the de-protonation of the B-hydrogens due to the acidification of the
responsible basic residue and the failure of the amine to rebound to the C3
position a required step to complete the synthesis of the B-product. The optimum
pH of 8.5 allows the substrate to be de-protonated off the ammonium proton
before it enters the active site, the free amine so generated and the carboxylate
provides the binding sites of the substrate to compliment the hydrophobic
interactions between the phenyl of phenylalanine and the leucine and isoleucine
residues (Figure 3.2). The argument in support of the aromatic ring as
nucleophile will be favored by low pH to ensure the ammonium form of the
substrate which will be prevalent to give way to the lost of ammonia to form the
Cinnamic intermediate. The ammonium form however will be difficult to maintain

under more basic conditions.

Site directed mutagenesis
The purified PAM mutants S176C, S176A and S176G were productive
with 4'-nitrophenylalanine, and 4'-flourophenylalanine which has electron

withdrawing groups (Figure 3.16 and 3.19). The 81760 mutant was also active

145

towards substrate with the natural substrate (Figure 3.16). The active of the MIO-
less mutants with substrates carrying electron-withdrawing substituents is
especially an interesting phenomenon and it explains the possible function of the
MIO during PAM catalysis. The nitro- or flouro- groups are believed to withdraw
electrons from the phenyl rind rendering the B-hydrogen acidic enough to be de-

protonated by a general base as shown in figure 3.24 below.

 

o
e ..
(SEN NH2
I
Ce
1 o
. e ..
O‘N/ NH2
l
06

Figure 3.24 The nitro group withdraws electrons from the phenyl ring causing the (3 hydrogen to
be acidic enough to be de-protonated by a general base. The same electron withdrawal is
proposed to apply with fluorinated substrates.

Both activity assays and differential spectroscopy evidence points to the
formation of the MIO in the S1760 mutant. The thio group could be hydrolyzed
the same as the serine of the ASG triad is in wild PAM. However the MIO yield in
the S176C might be less that in the wild type PAM (Figure 3.25). This aspect will .

need to be investigated further.

146

 

 

 

 

 

Scheme 3.25 The proposed formation of the MIO from the cyclization and condensation of the
ala-cys-gly triad. The reaction results in the loose of one water molecule and one hydrogen
sulfide molecules.

The use of crude S176G did not result in the formation of any j3- product
even with electron withdrawing substrates. Instead the corresponding D amino
acid is formed from the L isomer which is used as substrate. However the
reaction results in the formation of 4'-nitrocinnamic acid as determined by
UV/visible spectroscopy. It is not clear why the crude mutant will not from the B-
4'-nitrophenylalanine, it is most likely that the mutation reaction is in competition
with the phenylalanine racemase enzyme present in crude extracts from E. coli
or the B-product is formed below the detection limit. In the absence of the MIO in
mutant PAM and the presence of a racemase in the crude extract the reaction
may not favor the mutase activity but the racemase activity in which ammonia
does not rebound to the C3 position because it is not held in position by a

covalent bond which is the case in wild-type PAM (Figure 3.26).

147

 

Figure 3.26 The possible products formed from the reaction between crude wild-type PAM with
4'-nitrophenylalanine. The reaction is complicated by the presence of phenylalanine racemase
form E. coli which isomerizes D phenylalanine to the L isomer.

In wild-type PAM the MIO is involved in the elimination of ammonia from the a-
position and delivering it to the B-position as the trans-cinnamate intermediate
shifts or slowly moves out of the active site as is the case'with PALs in which the
release of the cinnamate from the site is the rate determining factor.101 However
the suggestion by Rétey concluded that in MIO-less HAL and PAL mutants the
ammonia may be held by coulombic forces may not be very plausible without
defining the origin of these forces and how specific they will be. The fact that the

HAL reaction is barely reversal may not be in support of such a mechanism. The

148

KM value of 4-NOz-L- o-phenylalanine and PAM reaction is ~1mM which is
significantly different from the natural substrate L- o-phenylalanine (45 pM). Our
KM is however similar to the one obtained by the Janssen group (1.8 mM). The

electron withdrawing of the nitro group might be acting syngestically with the six
positive helices which point towards the aromatic ring of the substrate as

suggested by Calabrese basing on the crystal structure of PAL from

Rhodosporidium toruloides.6' 52' 53

2, .
£21. ..

e222 #4"

N N\/

I.

Figure 3.27 Seven 0 helices associated with the active sites of PAL and HAL. Shown are the six
positive poles and one negative pole of the seven 0 helices, directed towards active-site residues,
with cofactor shown for reference.

Investigating the basic residue which deprotonates the B-hydrogen

The general base which de-protonate the [3 hydrogen is essential for the
PAM catalysis. In the absence of the base the reaction is greatly compromised
and decreases by more than 100 fold. The base not only does it act in removing

the proton, but it is believed to assist in shuttling to the free amine generated

149

when the first NHg-MIO complex breaks down. The created ammonium-MIO

complex donates a proton to the o-carbon of cinnamate intermediate to create a

carbocation of the (3 carbon of the alkene which is quenched by reacting with the

MIO-NH3 complex through 1, 4 Michael addition. The new intermediate
generated, C3'NH2-MIO complex is formed and the subsequent cleavage of the

NHz-MIO bond forms the B-acid and the free MIO to complete the catalysis. The

proton exchange between the tyrosine base and the amine explains why Mutatu
and co-workers reported a 30% transfer of deuterium from the C3 of the

substrate to the CZ position. It is envisaged that once the ammonium ion (MIO-
NH3) is formed there are equal chances (30%) of each of the three protons

adding to the 02 carbon of the cinnamate.

3.4 CONCLUSIONS

Phenylalanine aminomutase (PAM) from Taxus Canadensis carries a
catalytic moiety the 3,5-dihyro-methylidene-4H-imidazol-4-one (MIO based on
the cysteine inhibition results). In the absence of a crystal structure this evidence
places PAM in a family cofactor-less enzymes like ammonia lyases and other
aminomutases such as TAM from Streptomyces globisporous.15 PAM performs
best at pH 8.5 but tolerates more basic conditions than acidic conditions, which
supports the proposed mechanism in which a general base is involved in
shuttling protons during the reaction. PAM is over expressed best in E. coli cells

with rare codon optimization at temperatures lower than room temperature. The

150

lack of reactivity of Y322F is further evidence that NH2 is the nucleophile

because the general base has dual role in both de-protonating the [3 hydrogen
and setting up the elimination of the MIO-amine complex to form the cinnamate

intermediate, this warrant further investigation.

151

3.5 MATERIALS AND METHODS
3.5.1 MATERIALS

Complete, EDTA-free protease inhibitor cocktail tablets were purchased
from Roche Applied Science(Germany). Luria Bertani (LB) broth from Acumedia
and isopropyl-B-D-thiogalactopyranoside (IPTG) was purchased from Gold
BioTechnology (St. Louis, Missouri). All other chemicals were purchased from

Sigma-Aldrich.

3.5.2 PROCEDURES

3.5.2.1 PAM Expression Optimization and Purification

The pam plasmid (pam + pET14b) was purified from E.coli BL21(DE3) (
Stragene) cell cultures and was used to transform E. coli BL21(DE3) CodonPlus-
RlPL (Strategene). In brief, a bacteria colony was used to inoculate 5 mL of LB
media with ampicillin (100 pg/pL) and chloramphenicol (50 pg/pL) selection. The

cultures were incubated at 37 °C for 16 hours and used to inoculate 1 L of LB
and incubation continued until optical density (ODeoo) reached 0.6. Then IPTG (1

mM) was added and incubation continued at 16 °C for 16 hours.
The Culture was centrifuged at 5,000 x g for 20 min at 4 °C and the

supernatant was discarded. The pellet was resuspended in lysis buffer (50 mM

NaHzPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0, 2 tablets Complete protease

inhibitor per 20 mL of lysis buffer and 10 mg lysozyme), incubated on Ice for 30
min and then sonicated on ice at 1 minute pulses for 5 minutes with 1 min

interval in between pulses. The Iysate was centrifuged at 15,000 x g for 20 min at

152

4 °C and the pellet discarded. The semi-clear supernatant was centrifuged at
45,000 x g for 2 h at 4 °C, the pellet was discarded.

The cleared lysate was loaded to a pre-equilibrated nickel-nitriloacetic acid
(Ni-NTA) column (10 mL of 50% gel suspension) and mixed gently at 4 °C for 5
min and left to settle. The flow through was drained out and the resin washed

using 10 times column volume (150 mL) of wash buffer consisting of 50 mM

N8H2PO4, 300 mM NaCI, 20 mM imidazole, pH 8.0). The PAM was eluted with

elution buffer (50 mM, NaHgPO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The

elutions were assayed for PAM by SDS-PAGE with Coomassie staining.

3.5.2.2 Enzymatic Assay Techniques

The general enzymatic assay involved incubating the substrate (1 mM)
with PAM (10 pg — 5 mg) at pH of 8.5 in either 50 mM Tris-HCI or 50 mM
Phosphate buffer. The mixture was incubated in 31 °C water bath for time
periods ranging from 30 min to 24 hours. The reaction was quenched with 0.1 N
NaOH, to a pH of 11 before adding 5 equivalence of acetic anhydride or
ethylchoroformate, and reacted for 30 min at room temperature. The pH of the
solution was lowered to 2 with 1 N HCI and the acetylated amino acids extracted
with ethylacetate (3 x 2 mL). The organic layer was dried in a rotor vapor
resusupended in 100 pL methanol and titrated with tris

(methyl)siylyldiazomethane to a pale yellow permanent coloration.

153

3.5.2.3 Equilibrium Constant Measurements

To assess the equilibrium between product and substrate in the PAM
reaction, d-phenylalanine at 100 pM was added to 10 ml of PAM (at ~ 250
pg/mL) in 50 mM phosphate buffer (pH 8.0) and incubated at 30 °C. Aliquots (1
mL) were withdrawn from the reaction at various time points between 15 min-50
h and quenched by careful addition of 0.1 M sodium hydroxide, then the amino
acids were derivatized and analyzed by GC/EI-MS (Agilent Technologies,
California, USA). The relative amounts of product and reactant at equilibrium
were determined by linear regression analysis of the area of the base peak
fragment ion of the derivatized d- or B-phenylalanines (m/z 162 and 178,
respectively). Peak area was converted to concentration-of-product (or -
substrate) by solving the corresponding linear equation, which was derived by
plotting the area of the signature base peak ion, produced by the corresponding
authentic N-acetyI-amino acid methyl ester standard, against the concentration of

each standard in a dilution series ranging from 0 — 30 pM, (5 pM intervals).

3.5.2.4 The pH dependence of the PAM reaction

PAM was incubated with L-phenylalanine (1 mM) at pH values ranging
from 6.5. to 10.5 at a temperature of 31 °C for 3 hours (before reaching
equilibrium). The reactions were quenched and derivatized as described above.
In order to adjust for the buffering capacities at different pH two buffer systems:
pH 6.5 to 9.5, 50 mM Phosphate and pH 9.5 and above values a 50 mM sodium

carbonate buffer system was employed.

154

3.5.2.5 Cysteine Inhibition studies

The wild-type PAM and mutants were incubated with L-cysteine (10 mM)

and stirred for 1 h under aerobic conditions and absorbance measured at Amax

42,43

338 nm every 10 minutes or as described by R0ther et al except dithiothreitol

(1 mM) was added to keep the cysteine in reduced form.

3.5.2.6 Reversibility of the reaction

The reverse reaction by PAM was tested by incubating the enzyme (25
pg) with 1 mM of B-phenylalanine at 31 °C for an hour. The product was
derivatized as described before. The N-acetylated methyl ester was injected into
the GC-MS equipped with a chiral column (Varian, CP-ChirasiI-Val, 25 m x 5 pm

in diameter).

3.5.2.7 Kinetic isotope effect

In order to determine the rate determining step of the reaction a labeled
substrate ([3,3-2H]-phenylalanine and 100 pg of PAM in 1 mL of 50 mM

Phosphate buffer, pH 8.5) were incubated. The rate of reaction of the labeled
substrate was compared with the same concentration of unlabeled natural

substrate and an equivalent amount of enzyme.

155

3.5.2.8 Site-Directed mutagenesis.

The mutants (8176 to Ala, Cys, Gly and Thr) were constructed using
Strategene Quick-change XL Site-Directed Mutagenesis kit. The following
primers were synthesized by the MSU Genomics Technology Support Facility

(GTSF) for the following mutants;

Table 3.4 The primers used to construct mutants S176A, G, C and T, the mutated codons are in
bold underlined.

 

Mutant Primers

 

S176G Reverse; 5'-AG GTC TCC TCC AGC GCT CAC AGA TC-3’

 

FonNard; 5'-TG AGC GCT GGA GAC CTC ATC CCG CT-3'

 

S176A Reverse; 5'- AG GTC TCC AGC AGC GCT CAC AGA TC- 3'

 

Forward; 5'-TG AGC GCT GCT GAC CTC ATC CCG CT-3'

 

S176C Reverse; 5'- AG GTC TCC GCA AGC GCT CAC AGA TC- 3'

 

Forward; 5'-TG AGC GCT TGC GAC C TC ATC CCG CT-3'

 

 

S176T Reverse; 5'- AG GTC TCC CGT AGC GCT CAC AGA TC- 3'.

 

Forward; 5'-TG AGC GCT ACG GAC CTC ATC CCG CT-3'

 

 

 

The following mixture was put together for PCR;
10X reaction buffer (5 pL), pam plasmid template (10 ng, 0.3 pL), primers (125
ng each, 0.5 pL), dNTP mix (10 mM, 1 pL), Quick Solution (3 pL), dngO (39 pL)

and PfuTurbo DNA polymerase (2.5 U/pL, 1 pL).

156

The thermo cycle was set up as follows: segment 1, 1 cycle, 95 °C for 1
minute denaturing, 18 cycles at 95 °C for 50 s each, anneal at 60 °C for 50 s,
extend at 68 °C for 7 min, and further extension at 68 °C for 7 min. The PCR
product was digested with Dpnl for 1 hr, to digest all the methylated DNA

template plasmids.

3.5.2.9 Transformation of XL 10-Gold Ultra competent E. coli Cells.
The XL10-Gold® ultra competent E. coli cells (Strategene, USA) were

gently thawed on ice and a 45 pl aliquot was transferred to a pre-chilled 14-mL
BD Falcon polypropylene round-bottom tube. B-mercaptoethanol (BME, 2 pL)
was added to the cells and swirled gently, incubated on ice for 10 minutes and
swirled gently every 2 minutes. The Dpnl treated DNA (2 pL) was added to
aliquot of the ultra competent cells, swirled gently to mix and incubated on ice for
30 minutes. The tubes were pulse-heated in a 42 °C water bath for 30 s,
incubated for 2 minutes and pre-heated LB (0.5 mL) added to each tube, then
incubated the tubes at 37 °C for 1 hour with shaking. The cells (250 pL) were

spread on ampicillin (100 pg/L) plates and incubated at 37 °C for > 16 hours.

3.5.2.10 Making Agar plates
To Luria Bertini (LB) (25 g) was added 20 g of agar and M0 H20 to a final

volume of 1 liter. The mixture was autoclaved for 30 min and cooled to 55 °C

before adding 1 mL of 50 mg/mL chloromphenicol and 1 mL of 100 mg/mL

157

ampicillin. The agar was poured (30 mL) into each plate and gently swirled to

make even. The solidify plates were wrapped and stored at 4 °C.

3.5.2.11 Purification and sequencing of Plasmid

Colonies (BL21-CodonPlus) from transformations were transferred to 5
mL LB media treated with ampicillin (100 pg/mL) and grown overnight at 37 °C.

The cells were centrifuged at 5,000 x g for 12 min. The supernatant was
discarded and the plasmid purified from the cells following the 0iagen Protocol.
The plasmids were quantified using the UV method by measuring the
absorbance at 260 nm and comparing with absorbance at 280 nm. Sequencing
was done at the MSU Genomics Facility and using forward primers and reverse

primers (Table 3.5).

Table 3.5 Primers used to sequence the pam ene

 

Forward primers Reverse primers

 

T7 promoter; 5'-TAA TAC GAC TCA T7terminator; 5'-GCT AGT TAT TGC

 

500 bp; 5'-TGC CTC TCC GAG GAT 500 bp; 5'-CAG GCA CCG C'IT TGG

 

1000 bp; 5'-TGG TGC AGA CAA TCA 1000 bp; 5'-GGA GAG CCC TGT

 

1500 bp; 5'-GAA GCC CTA GTA AAA 1500 bp; 5'-GCC TAT GCG AGC

 

 

2000 bp; 5'-GCC TCT GCT GCA CTG 2000 bp; 5'-GGT CGT ACC GTC TAC

 

 

 

158

3.5.2.13 Culture Conditions.
E. coli BL21CodonPlus -RIPL cells were used to over express both wild
type and mutant PAMs. Cells were grown in 1 liter Miller LB medium

supplemented with ampicillin (100 pg/pL) and chloromphenicol (50 pg/pL) initially

at 37 °C. At an ODeoo of 0.6, 1 mM of lsopropyl B-D-thiogalactoside was added,

the temperature reduced to 16 °C and incubation continued for 16 hours.

3.5.2.14 Comparison of Kinetic Characteristics of Wild- Type PAM, the mutants
S176A, S176G, 8176T and 81760 with 4'-nitro-L-Phenylalanine and 4'-F-
phenylalanine as substrates.

The kinetic measurements were determined by GC-MS analysis, HPLC
and uv-visible spectrophotometer. The substrate concentrations were 0.25, 0.5,

1, 2, 3, 4, 6, 8 and 10 mM. The concentrations of product were plotted on Excel

(Microsoft 2007) by fitting all initial rate data to the Michaelis-Menten equation
using nonlinear regression (R2: 0.99). The assays were carried out as described

above.

3.5.2.15 Spectroscopic studies of PAMs
The difference absorption spectra of the PAM mutants were measured by
scanning from 240 to 360 nm. The mutants S176G, S176A, 81760 and S176T

were used as blanks, against the wild-type.

159

3.5.2.16 Construction of the mutants to investigating the basic residues

The mutants Y322A, H, and Y and C107A and H were constructed
following the Strategene Quick-change XL Site-Directed mutagenesis kit. The
following primers were synthesized by the MSU Genomics Technology Support

Facility (GTSF) as shown in table 3.6;

Table 3.6 The primers used to construct mutants Y322A,H and F, and 0107A and H; the mutated
codons are in bold underlined.

 

Mutant Primers

 

Y322A Reverse; 5'-CT CAG AGC ATA GCG ATC CTG TIT CG-3'

 

Forward; 5'-AG GAT CGC TAT GCT CTG GGT CGA GC-3'

 

Y322H Reverse; 5'-CT CAG AGC ATG GCG ATC CTG TI'T CG-3'

 

Forward; 5'-AG GAT CGC CAT GCT CTG GGT CGA GC-3'

 

Y322F Reverse; 5'-CT CAG AGC GAA GCG ATC CTG T'l‘l' CG-3’

 

Forward; 5'-AG GAT CGC TI'C GCT CTG GGT CGA GC-3'

 

C107A Reverse; 5'-CG GAG CAG TGC GCG TAT AAG CGA CT- 3’

 

Forward; 5'-TT ATA CGC GCA CTG CTC GCG GGG GT-3'

 

 

C107H Reverse; 5'-CG GAG CAG ATG GCG TAT AAG CGA CT- 3'

 

Forward; 5'-Tl' ATA CGC CAT CTG CTC GCG GGG GT-3'

 

 

 

PCR protocol: The following mixture was put together; 10X reaction buffer (5 pL),
pam plasmid template (10 ng, 0.3 pL), primers (125 ng each, 0.5 pL), dNTP mix
(10 mM, 1 pL), Quick Solution (3 pL), dngO (39 pL) and PfuTurbo DNA

polymerase (2.5 U/pL, 1 pL).

160

The thermo cycle was set up as follows; segment 1, 1 cycle at 95 °C for 1
minute denaturing, 18 cycles ( at 95 °C for 50 s each, anneal at 60 °C for 50 s,
extend at 68 °C for 7 min) and further extension at 68 °C for 7 min. The PCR
product was digested with Dpnl for 1 h, to digest all the methylated plasmids

including the DNA template.

3.5.2.18 Growth and harvest of mutant enzymes
The mutants Y322 and 0107 were grown and harvested as described above for

wild type PAM.

3.5.2.19 Activity Assay

The activities of the mutants were performed as described for the wild-

type above.

161

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171

CHAPTER 4

Recombinantly Expressed Phenylalanine Aminomutase (PAM) Shows
Increased Activity In E. Coli Grown Under Light Illumination

4.1 INTRODUCTION

Light illumination has been implicated in shifting the redox state of
bacterial, yeast and mammalian cells and activating the synthesis of ATP and
DNA,"2 hence stimulating the synthesis of both recombinant and non-
recombinant proteins. This photobiological reaction involves the absorption of a
specific wavelength of light by photoreceptors which are not normally involved in
light responses such as in photosynthetic organisms (e.g. chlorophyll). The
photoreceptor must be a key structure that can regulate a metabolic pathway in
the organism. Examples of such receptors are redox chains such as

cytochromes and flavoproteins. Evidence has shown that mitochondria are
sensitive to irradiation with monochromatic visible and near-infrared light.3 The

light response has been observed in both bacterial (E. coli) and mammalian cells

in which there is increased ATP synthesis and 02 consumption?5 Light
irradiation, increases the mitochondrial membrane potential, proton gradient,
Optical properties, rate of ADP/ATP exchange and a shift in the NADH/NAD,
NADP/NADPH, and GSH/GSSG ratios.6 The respiratory chain of mitochondria as

represented by cytochrome c oxidase presents a fulcrum into the link between
the light activation and the change of the redox potential. The enzyme contains

metal ion centers which are prone to photoredox reactions and has influence on

172

the bioenergetics of the cell. Different wavelengths of light will give different and
sometimes antagonistic responses on the cellular metabolism, for example red

light at 650 nm has been reported to increase oxidative phosphorylation, but far-
red at 725 nm inhibited it,5 which point to the absorption of different

chromophores which are in different redox states and are involved in different
metabolic pathways. In brief the terminal respiratory chain oxidases in eukaryotic
cells (cytochrome c oxidase) and in prokaryotic cells (cytochrome bd and b0
complexes) are believed to be photoacceptors molecules for red-to-near-IR

radiation and the flavoproteins (NADH dehydrogenase) are response to the
violet-to-blue light.1 The irradiation of cytochromes with light shifts to more

reduced form thus promoting electron transport which may also trigger folding of
the proteins. Apart from the electronic effects of the chromophores and
photosensitive proteins, there is a possibility that irradiation may increase
temperature of the biomolecules which may cause structural (conformational)
changes and trigger biochemical activity (secondary dark reactions) such as

activation or inhibition of enzymes. The increased electron flow in the respiratory

chain, increases the superoxide anion (02") production, which provides a source

of electrons for the oxidative phosphorylation of ADP under physiological

conditions.7 The related increase in H202, results in multiple responses such as

increase in; [Ca2+], pH, activation of Na+/H+ antiport (involved in the photosignal
transduction and amplification chain), Ca2+ ATPase, Na+, K+ ATPase (which

control CAMP levels in the cell) and alteration of NaJ'-Ca2+ exchange.8'9 Catalase

173

the enzyme involved in the conversion of 02" to H202 has been observed to

increase in activity in yeast cells.10 The absorption of light (visible) by certain

porphyrins and flavoproteins generate singlet oxygen which act as a mediator in

11-23

achieving the biological effects of radiation. Trushin demonstrated that light

can increase the expressron of a recombinant protein barstar in E. coir. 9 23

The most extensive studies on the effect of light on living cells is provided
by the plant systems. Plants have been reported to respond to environmental

24-72

factors such as heat, light and predators. The response of plants to such

external stimuli may change the metabolic pathway by regulating expression of
enzymes or synthesis of special metabolites which may act to protect the plant.
The studies on plant systems has been focused on the phenylpropanoid
pathway, which is initiated by phenylalanine ammonia lyase (PAL) and/or
tyrosine ammonia lyase (TAL). PAL and TAL are involved in the non-oxidatively

de-amination of phenylalanine and tyrosine to trans-cinnamic and p-coumaric

73-1 00

acid respectively. Hahlbrock and co-workers observed that plant cell

cultures of Petroselinum hortense had a marked increase in phenylalanine

ammonia lyase (PAL) activity when grown under light illumination versus dark

101-105

conditions. Their results suggest that light increases both the de novo

synthesis of PAL and its activity. Speculations about the effect of light on the
enzymes has been implicated on the influence of light, especially UV on trans-
cinnamic acid which is photochemically transformed to Cis-cinnamic acid. The

depletion of trans-cinnamic to Cis-cinnamic acid will probably cause a negative

174

efflux of trans-cinnamic acid and hence increase the activity of the

106 Similar studies on histidine ammonia

phenylpropanoid pathway enzymes.
lyase (HAL) which catalyzes the conversion of L-histidine to urocanate have
indicated a positive response to light. PAL and TAL are structurally and
mechanistic similar to phenylalanine aminomutase (PAM) which converts a-
phenylalanine to B-phenylalanine. Not only do these enzymes share a 56%

homologY. the possess 12 conserved residues and have a catalytic moiety 3,5-

dihydro-S-methylidene imidazolone-4-one (MIO) which is implicated in the

107-129

mechanism of catalysis. The cofactor is auto catalytically and post

transitionally synthesized by the cyclization and dehydration of the alanine-

130

serine-glycine (ASG) triad. Yu and coworkers demonstrated using mass

spectrometry that a high proportion (90%) of the TAL protein is in the MIO-less
hence inactive form. The light illumination could be increasing the MIO per
protein ratio thereby increasing the active form of the enzyme relative to the total
enzyme. The light regulation could still be attributed to the phenomena described
above related to the shift in the redox potential of the cell, which has effect of
phosphorylation of enzymes.

The study of the phenylpropanoid provides a direct link between
increase in protein expression and activity. Interestingly the bacterial and plant
metabolic pathways are linked by the phenylpropanoid pathway (Figure 4.1).
Specifically the ubiquitous plant enzymes phenylalanine ammonia-lyase (PAL)
and chalcone synthase (CH8) are key biosynthetic catalysts in phenylpropanoid

and flavonoid assembly, respectively. Bacteria such as Streptomyces maritimus

175

and E. coli produce benzoyl-00A in a plant-like manner from phenylalanine
involving a PAL-mediated reaction through cinnamic acid during the biosynthesis
of the polyketides. The recent discovery that bacteria harbor homodimeric PKSs
belonging to the plant CHS superfamily of condensing enzymes has further
linked the biosynthetic capabilities of plants and bacteria suggesting a similar
mechanistic response to light irradiation. If the light activation is specific to the
phenylpropanoid enzymes, which is arguable since some mammalian enzymes
have been reported to be responsive to light, then it may point to the fact that all
enzymes are activated by light, whether they are recombinant or not. It follows
then that an enzyme like PAM which is heterologously expressed in E. coli
should be activated by light through any of the mechanisms described above.

In this study we focus on measuring both the expression and activity of
PAM. The activity of PAM will be measured by the rate of B-phenylalanine
formation in vivo and the results compared with a control samples kept under
non-illuminating conditions. An attempt will be made to assess whether the
observed increase in B-phenylalanine formation under light illumination is
attributed to the post-transcriptional modification of PAM or the stimulation of the
biosynthetic pathways of E. coli through the mechanisms explained above. The
variation of PAM activity over time in response to light illumination and the effect
of light on the growth and cell viability as measured by cell density (optical
density at 600 nm) of transformed bacteria is also reported. The effect of light on
the thermal activation via increase in temperature of the medium was addressed

by monitoring the temperature over time. The type of radiation responsible for

176

PAM activation was determined by irradiating the cell cultures with white light
filtered from red and/or IR radiation. The effect of sonication on the activity of
PAM was investigated and also the effect of visible light and ultra-violet light on
the activity of pure PAM was studied. If indeed light increases activity of PAM
and other enzymes it will provide an easy and inexpensive way of producing
more efficient enzymes per gram of cell pellet without increasing the amount of

cell cultures.

0
©/u\OH , OH

Phenylalanine racemase

O 0 O
\ , Oxidative deaminase
OH 1 Ammonia lyase OH = OH
NH2 0
0
or”:

0

Z
I
N

_—Ié

 

 

Decarboxylase

NH2

3

Figure 4.1. A scheme showing the fate of L-phenylalanine in E. coli. The presence of PAM in E.
coli will increase the flux of trans-cinnamic acid thereby upsetting the cellular metabolism which
may contribute to different response to light conditions.

177

4.2 RESULTS AND DISCUSSION
4.2.1 Results
4.2.1.1 Assaying for PAM activity

The activity of recombinant PAM in cells exposed to total white light was of
3.8 1 0.5 % at 16 °C (Figure 4.2) and 18 :l: 3% at 25 °C as measured by the
amount of endogenous 0 -phenylalanine converted to B-phenylalanine. The cells
kept in the dark had an activity of 1 :1: 0.5% (Figure 4.2) and 10 :l: 2% at 16 °C
and 25 °C respectively. The activity was measured as the mole fraction of (R)-B-
phenylalanine relative to the total phenylalanines in the culture. The cells were
lysed chemically and the amino acids derivatized to their N-acetylated and
methyl ester form and analyzed using GC-MS. PAM is usually harvested after
IPTG induction at 16 °C, 18 °C or 25 °C and incubation for 18 h. The optimum

temperature is 16 °C as reported and discussed in Chapter 2. The optimum

temperature for PAM activity is 28 °C.131

178

P
01

 

A
-—I

 

99
01
L

0)
1

PAM Activity (% beta per g of cell pellet)
.. N
-s in N at

p
0|
1

 

 

 

 

 

 

LIGHT DARK

Flgure 4.2 Effect of total white light illumination on phenylalanine aminomutase activity in E. coli

at 16 °C. The amino acids were derivatized as described above and assayed by GC/MS. The
light illuminated had an average activity of 3.8% per gram of cell pellet and the non-illuminated
had about 1% (black).

4.2.1.2 Effect of lysis technique on PAM activity measurement under light
illumination conditions

The cells from the illuminated and covered control experiments were lysed
by either sonication or by chemical lysis using a series of buffers. The sonicated
set of samples (Figure 4.3), DSL (covered and Iysed by sonication) and LSL
(illuminated and lysed by sonication) there was no significant difference between
light illuminated and non-illuminated conditions whereas for the chemically Iysed

set of samples showed a 6 fold difference.

179

 

 

 

 

 

 

 

 

 

 

 

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5
66“
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Q.
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E
5°2—
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DSL DCL LSL LCL

 

 

 

Figure 4.3 Effect of method of lysis on PAM activity measurement -Cells illuminated by total white
light or grown under non-illumination conditions were either lysed by sonication (DSL and LSL) or
chemically (DCL and LCL).

DSL- sample covered (dark) and Iysed by sonication

DCL- sample covered (dark) and lysed chemically

LSL- illuminated by light and lysed by sonication

LCL-illuminated by light and lysed chemically

4.2.1.3 Investigation of the effect of UV light on the activity of pure PAM.

To investigate whether the increase in PAM activity when exposed to light
was due to activation of the expressed enzyme, pure PAM was incubated with
substrate and exposed to UV-B (254 nm) at room temperature for 1 hour,
alongside a control covered in aluminum foil. The UV exposed reaction light
showed a less that one percentage point increase in activity compared to the
control (Figure 4.4). UV radiation has a small contribution to the activity of pure

PAM. The small increase could perhaps be due the activation of the exocyclic

180

 

double bond of the methylidene imidazolone (MIO). The slight increase in B-
phenylalanine formation could also be attributed to thermal effects which could

cause an increase in the local temperature.

 

60

 

50:

40*

30~

20:

PAM activity (% mol B-phe)

10~

 

 

 

 

 

0 ' ' . ' I . .
uv 1h RM light 1h Dark 24h Light 24h

 

 

 

 

Figure 4.4 The effect of UV radiation (254 nm) on the activity of pure PAM, incubated with 1 mM
of a-phenylalanine at room temperature for 1 h and 24 h respectively. There is a slight increase
(0.1%) in activity under UV light illumination (A) than room temperature light (B). The samples
exposed to light (120, 65 W light bulb) but covered in aluminum foil (0) and an uncovered (D) and
incubated overnight showed no significant difference.

4.2.1.4 Effect of sonication and light illumination on pure PAM

The effect of sonication was investigated by running 4 parallel reactions
one in which a purified PAM-substrate mixture was sonicated for 5 min (1 min
pulse, 1 min intervals) in ice; the second sample was incubated at room
temperature under total white light illumination; the third sample was covered in

aluminum foil and incubated at room temperature and the fourth sample was

181

incubated in ice for 10 min. The control sample kept in ice for 10 min had no
activity. The sonicated sample had 4 % activity and the two samples kept at room
temperature under dark and light illumination conditions respectively had 30 %

activity (Figure 4.5).

 

3.5

1

 

2.5 .

1.5 J

 

I"!

PAM activity (% mol B-phe)
M

0.5 ~

 

 

 

 

 

 

 

 

 

Ice Sonication Light 1h Dark 1h

 

 

 

Figure 4.5 The effect of sonication on the activity of pure PAM. Samples kept in ice (ICE) showed
no activity. Samples sonicated for a total of 5 minutes had 1.4 % mol conversion. Control samples
incubated for 1 hour at 31 90 under light illumination and dark/covered had about 3.2 % mol
conversion.

4.2.1.4 Effect of pre-sonication on the activity of pure PAM

The cell pellets Iysed by sonication did not show any difference in activity
between light illuminated and dark/covered samples (Figure 4.6). Sonication
seems to allow the covered samples to ‘catch up’ with that exposed to light. Its
either the sonication increases the rate of reaction by improved intermolecular

collisions or there is an increase in temperature which accelerate the reaction.

182

Sonication could also be allowing the refolding of the protein and maybe able to
form the more active form of the enzyme possibly via additional MIO formation. In
order to verify this phenomenon pure PAM was sonicated first before incubating
with substrate. If sonication increases amount of active enzyme then an
unsonicated sample will have less activity. The results in Figure 4.6, shows a

slight increase in PAM activity in the sonicated sample.

 

 

 

 

 

 

 

 

 

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<6
2 4 ~
:5

2 1
0 .
Sonication Control

Flgure 4.6: Shows the effects of sonication on PAM activity. PAM was first sonicated for 5 min
before addition of substrate and incubation for 1 hour. The control was incubated for the same

time and temperature and time without sonication.

183

4.2.1.6 Time course assay

Time course assay shows a time dependent variation of PAM activity

which peaks between 6 to 8 hours after IPTG induction at 25 °C (Figure 4.7).

 

 

 

 

 

 

 

 

 

 

55
e 45 - i
g —o— DARK I
<5- 35 - - -I- - LIGHT ,'
6 5 .
E ;
°\°
.E‘
.2
‘5
<6
2
<
O.
O 5 1O 15 20 25 30
Time(h)

Figure 4.7 Shows the averaged time course assay trend of the variation of PAM activity under
light illumination (square) and non-illumination (rhombic).

4.2.1.7 Monitoring of E. coli growth illuminated with light filtered from red and/or
IR radiation

The growth of E.coli was monitored over an 18 hour period by measuring
the cell density at a wavelength of 600 nm. The uncovered sample exposed to
total white light appeared to reach the saturation phase earlier than the covered

cultures or where a portion of the spectrum was cutoff (Figure 4.8).

184

 

 

 

3-5 ‘ -+-covered + IR cutoff
3.4 1
3.2 2 +uncovered + IR cutoff
3 4 -A1- covered + IR and red
g 2.8 - light out off
8 2.6 1 -><° ~uncovered + IR and
2.4 1 red Iightcutoff
-->I<- covered
2.2 1
2 2 n... uncovered
1.8 . T . .
7 9 11 13 15

Time,h
Figure 4.8 Monitoring the growth of E.coli cells transformed with the pam gene under various

light conditions. The cultures illuminated with total white light without any filters had higher

ODsoo (.) before the saturation phase. The covered cultures and those exposed to light in which
the red and/or IR radiation had been cutoff showed reduced growth during the log phase.

4.2.1.7.2 Effect of Different Types of Radiation on the Activity of Recombinant
PAM Grown in E. Coli.

A comparative study of the effects of red, infrared and visible light on the
activity of PAM in E. coli was conducted by having; 1) a set-up in which red and
infrared radiation had been eliminated, 2) only the infrared radiation was used to
illuminate the E.coli, and 3) total white light ( + IR) was used to illuminate the
samples. Each set-up was run parallel to E. coli cultures which were covered in

aluminum.

185

 

 

 

 

 

 

 

 

 

 

3500 1———-———~—
3000 —o - covered + IR cutoff
g filter
3 2500 .7 * -l - uncovered + IR cut off
g 2000 ’Jffv; .. - 1 filter
gt -' —*—covered + RED and IR
'5 1500 cut off filter
ch. 1000 - «x- uncovered + RED and
at
1 IR cut off flter
500 - X- covered + total white
0 light
7 9 1 1 13 15 no» ltiincovered + total white
ght
time, h

 

 

 

Figure 4.9 Comparative assays to study the effects of red light, infrared and visible radiation on
the activity of PAM expressed in E. coli. All the uncovered samples had higher activity than the
covered ones, with the uncovered and unfiltered giving the highest values.

4.2.1.8 Identifying phosphorylation sites in PAM

Peptide sequencing showed less than 12% probability of phosphorylation.
Phosphorylation sites are usually on serine residues. The trypsin digested PAM
was assayed by LC/MS/MS and the peaks searched against all bacterial and
green plant protein sequences and confirmed the presence of phenylalanine
aminomutase from T. Canadensis. The Mascott output analyzed by protein
computer algorithm at 95% probability did not show any phosphorylation. Only

two sites were identified but the probability was below 12% for both of them.

186

4.2.2 DISCUSSION

The exposure of E. coli transformed with the pam gene plasmid to
continuous light produced the mutase with more than 3 fold increase in activity
compared to the dark (Figure 4.2). The exposure of light to purified PAM does
not increase activity significantly compared to samples kept in the dark (Figure
4.4). The observed increase in activity of PAM expressed in E. coli could be due
to several possible phenomenon ranging from the perturbation of the bacterial
metabolism to the production of more active enzyme possibly influenced by
oxidative phosphorylation and/or increased MIO formation. E. coli metabolic rate
has been observed to be increased by light illumination particularly visible, IR
and red light through the increased production of DNA and ATP resulting from
the retrograde process in which the mitochondria are bio-stimulated. The photo-
stimulation causes increase in cell proliferation and upregulation of genes and
hence heightens the synthesis of both non-recombinant and recombinant
proteins. Although we could not quantitatively measure the amount of proteins
produced under different light conditions the cell density offers an approximation
of the cellular composition and indication of increased metabolic rate. The cell
density measurements shows that light illumination seem to accelerate the rate
at which E. coli reaches the saturation phase (Figure 4.8]. The observed
difference in cell density between illuminated and non-illuminated cells does not
correspond to the difference in the reactivity of the enzyme (PAM), which
suggests that the mechanism of the increased activity under light could not be

related to cell growth and may be specific to PAM as an enzyme observed with

187

homologous enzymes such as phenylalanine and histidine ammonia lyases

132-173

which have been demonstrated to be activated by light in plants. These

enzymes have a characteristic MIO cofactor which also is also present in
phenylalanine aminomutase (PAM). It appears like the increase in PAM activity
could be de novo and reaches a maximum limit before it levels off or decreasing
(Figure 4.7), similar to observations made on PAL. The regulation of PAM by light
likely occurs in vivo suggesting the involvement of cellular transformation either
through phosphorylation, synthesis of higher amounts of the MIO and possibly
the decrease in the trans-cinnamic acid flux attributed to its photochemical
conversion to its Cis-isomer. The lack of response of pure PAM suggests the
regulatory factors which increase its activity are de novo. The expression of PAM
based on SDS-PAGE with Commassie blue staining did not show any significant

difference between light and dark samples.

Effect of phosphorylation

Phosphorylation and de-phosphorylation have been implicated in
stimulating PAL expression and activity in tomato seedlings and Parsley cell
cultures. Similar mechanism could be happening to PAM, regulated through the
mitochondrial photoactivation and the effect on the respiratory chain. However no
phosphorylation could be confirmed in our study following trypsin digestion and
LC/MS/MS analysis. The involvement of kinases in PAL activation has not been
conclusive due to the different iso-forms of PAL (PAL I, II and Ill). If

phosphorylation decreases PAL activity, then de-phosphorylation of pure PAM

188

 

should reverse the loss of activity. There is a likely would that the formation of the
MIC could be related to phosphorylation since phosphorylation requires serine or
threonine and a serine is involved in the formation of the MIO. Once the protein is
phosphorylated no MIO could be formed, since phosphorylation will block MIO
formation. De-phosphorylation will free up the serine for MIO formation hence
increased activity. However LC/MS/MS results have shown no phosphorylation
on the serine making up the ASG triad which makes the MIO. The evidence of
PALIl phosphorylation could not be proved by immuno-assay techniques. Further
studies need to be done to confirm phosphorylation in PAM, by both

immunoassay and mass spectrometry.

MIO formation

The mechanism by which the MIO is auto-catalytically formed in PAL or
PAM is not yet understood, but similar synthetic process has been observed in
the green fluorescent protein (GFP) found in Aequorea Victoria bacteria in which
serine, tyrosine and glycine cyclize followed with an oxidative process. Further
investigation need to be carried out to verify whether there is an increased, in
MIO formation under light conditions versus dark conditions. Mass spectrometry
experiments with TAL have shown that, not all enzyme molecules carry the MIO
motif. In fact as much as over 90% of TAL could be in the unmodified form. Our
LC/MS/MS results did not confirm the presence of the MIC moiety in PAM

probably due to its low concentration, yet the ASG triad was abundant. There is

189

need to quantitatively compare the amount of MIO synthesized under light

illumination and non-illumination conditions.

The trans-cinnamic Feedback mechanism
Another perspective to the increase in activity under light illumination
conditions is the perceived feedback mechanism in which trans-cinnamic acid is

isomerized to Cis-cinnamic acid (Figure 4.10), a reaction that is accelerated by

light.174

More trans-cinnamic would have to be made thus up regulating the
phenylpropanoid pathway. Alternatively the trans-cinnamic acid formed from the
heterologously expressed PAM reaction may be pertubing the bacterial
metabolism by increasing the activity of the enzymes downstream of the

cinnamic acid pathway (Figure 4.2).

 

 

O h H H
v _
. O
trans-cinnamic acid Cis-cinnamic acid

Figure 4.10 The conversion of trans-cinnamic acid to Cis-cinnamic acid under ultraviolet light.

PAM expression could be regulated by trans-CA conversion to Cis-CA and
hence change the ratio of a to B-phenylalanine in the cell. Trans-cinnamic acid
has been observed to inhibit PAL at pH ranging from 8.3 to 9.3 which falls within
the optimal pH for PAM (Chapter 2). NMR kinetic studies have shown that the
conversion of fi-phenylalanine to trans-cinnamic acid is much faster than a-

phenylalanine to the same product (Chapter 5). The depletion of t-CA would off-

190

set the equilibrium between q-phenylalanine, trans-cinnamic and B-phenylalanine
is shown below (Figure 4.11 ), resulting in less B-product being synthesized

which is contrary to our findings, thus ruling out the t-CA flux theory.

ing2 0 o 0
OH --—-- \ OH OH
NH2

Flgure 4.11 The equilibrium between B-phenylalanine, trans-cinnamic and a-phenylalanine

 

 

 

Effect of sonication and heat on PAM activity

Sonication increases the activity of PAM both in vivo and in vitro (Figure
4.5 and 4.6). The mechanism may not be clear but its most likely through thermal
activation and increased vibrational motions. However an interesting
phenomenon is the increased activity of pre-sonicated PAM (Figure 4.6).
Perhaps sonication causes the re-folding of the enzyme into an active form or

speculatively may form more of the catalytic moiety (MIO).

191

4.4 CONCLUSION

The presence of light during de novo synthesis of PAM regulates its
expression and activity in a fluctuative manner in which activity increases during
the initial growth phase and decreases at later stages. It’s not clear how the
regulation happens but it’s likely that light affects the synthesis of the catalytic
moiety MIO which is directly linked to the mechanism of PAM and similar
enzymes. The perceived light illumination regulation will open up an opportunity
to attenuate MIO based enzymes expression and activity. Increasing the activity
of enzymes will overcome solubility challenges encountered during protein
expression and purification. There is still remaining a possibilty that light could be
influencing the protein expression in E. coli by tweaking the redox potential of the
cell resulting in increased metabolic activity.

Further investigation need to be carried to confirm the relative amounts of

MIO formed under light illumination and non-illumination conditions.

192

4.5 MATERIALS AND METHODS
4.5.1 Instruments and Reagents

All reagents were purchased Sigma Aldrich - unless otherwise noted and
used without further purification, except for 3'-methylphenylalanine which was

purchased from Peptech (Burlington, USA), 4'-nitrocinnamic acid from Fluka

(Steinheim, Germany). Buffers P1, P2 and N3 were sourced from the QlAprep®

Spin Miniprep Kit-250 (Qiagen, Maryland, USA). IR and red light Cut-off filters
(50 mm x 50 mm x 3 mm) were purchased from CV Melles Griot (Rochester,
USA). The light emission wavelength spectrum was measured on an Ocean
Optics instrument; model USB 2000+ (Dunedin, Florida). Radiant power was

measured with a Nova ll instrument from Ophir (Jerusalem, Israel).

4.5.2 EXPERIMENTAL PROCEDURES
4.5.2.1 General assay of amino acids in cell cultures

At each sampling time point, the cell cultures were harvested and
centrifuged at 4,500 g for 20 min at 4 °C. The supernatant was basified to pH 10
(3 M NaOH), acetic anhydride (500 pL) was added to each sample, and the
reaction mixed for 10 min at room temperature (25 °C). The pH was decreased to
2 (3 M HCI) and the N-acetylated amino acid was extracted with ethyl acetate (2
x 3 mL). The ethyl acetate was evaporated, and the residue was resuspended in
100 pL methanol and trimethylsilyl diazomethane solution in diethyl ether was
added until the yellow color of diazomethane persisted. The solvent was
evaporated, and the residue re-dissolved in ethyl acetate (250 pL) and analyzed

by gas chromatography (model 6890N, Agilent, Palo Alto, CA) coupled to a mass

193

 

selective detector (model 5973 inerl®, Agilent) equipped with a 5HS GC column
(0.25-mm inner diameter x 30 m, 0.25-pm film thickness) mounted in the GC
oven. The MS conditions were set with an ion scan mode from 100 — 300 atomic
mass units and the ionization voltage was set to 70 eV. The (30 conditions were
as follows: column temperature was programmed from 70 °C (3 min hold) to 320
°C at 10 °C /min and then a 3-min hold at 320 °C, splitless injection was selected,

and helium was used as the carrier gas at a flow rate of 1.2 mL/min.

4.5.2.2 Over Expressing PAM under continuous white light illumination conditions

E. coli BL21CodonPlus (DE3-RlPL) colonies transformed with the pam
gene sub-cloned into pET14b vector were used to inoculate 5 ml of Luria Bertani
(LB) media containing 100 ug/pL of ampicillin and 50 pg/uL of chloromphenicol
antibiotics. The tubes were wrapped in aluminum foil to block light and incubated

at 37 °C overnight. The 5 mL cultures were transferred to a 2.8 L flask containing
1 L LB media and covered with aluminum foil and incubated at 37 °C until ODsoo

was about 0.6. The 1L culture was induced with 1 mM lsopropyl-D-
thiogalactopyranoside and divided into two, one of the flasks covered in
aluminum foil, and incubated at 16 °C or 25 °C with light illumination from a 65 W,

120V bulb (Maximum overall length, 770 lumens, 100 CRI, Spot-Gro Sylvania,

light intensity of 0.14 W/cmz) for 18 h. The cell cultures were divided into 50 mL

portions and centrifuged at 4,500 x g for 30 min. The supernatant was discarded

and the cell pellet treated as above.

194

4.5.2.3 Monitoring of trans-cinnamic acid

The cell cultures were centrifuged as above except that the pH was
reduced to 2 before extracting with EtOAc. The organic layer was dried and the
residue dissolved in methanol before adding diazomethane solution and reacted
for 30 min. The solvent was evaporated in a speed vacuo and the residue

resuspended in EtOAc and analyzed by GC/MS.

4.5.2.4 Purification of PAM
E.coli BL21 Codon plus (DES-RIPL) cells were grown in 1 liter Miller LB

medium supplemented with ampicillin (100 ug/pL) and chloromphenicol (50

ug/uL) at 37 °C until 00500 of about 0.6 and induced with 1 mM IPTG. The

temperature was reduced to 16 °C or 25 °C and incubation continued for 16
hours or longer. The culture was centrifuged at 7,500 x g for 20 min at 4 °C and

supernatant discarded. The pellet was resuspended in lysis buffer (50 mM

NaHgPO4 pH 8.0, 300 mM NaCl, 10 mM imidazole, 2 tablets (Complete, Roche,

German), protease Inhibitor and 10 mg lysozyme, incubated on ice for 30 min
and then sonicated on ice at 1 minute pulses for 5 minutes. The lysate was
centrifuged at 15,000 rpm for 20 min at 4 °C and the pellet discarded, the semi-
clear supernatant was further clarified at 45,000 rpm for 2 h, the pellet was

discarded and the supernatant processed as below. The cleared lysate was
loaded onto a pre-equilibrated (50 mM N3H2PO4 pH 8.0, 300 mM NaCl, 20 mM

imidazole) Ni-NTA column and mixed gently at 4 °C for 5 min and left to settle.

The flow-through was drained out and the column washed with 10 times column

195

volume of wash buffer consisting of 50 mM NaHgPO4, 300 mM NaCl, 20 mM

imidazole, pH 8.0. The PAM was eluted with elution buffer (50 mM, NaHzPO4,

300 mM NaCl, 250 mM imidazole, pH 8.0) and the fractions were assayed for
PAM using SDS-PAGE with Coommassie Blue staining as described elsewhere.
The fractions containing pure PAM were pooled together and concentrated by
ultra filtration through an Amicon 50 kDa membrane (Centriprep® Centrifugal
Filter Units 50,000 MWCO, Millipore, Billerica, MA) at 4 000 x g to 1mL final
volume. The PAM content was analyzed using the Bradford method (9) using
BSA as the standard. The enzyme with a purity of over 95 % was kept at -80 °C

(after adding 5% glycerol) until required.

4.5.2.5 Investigation of the effect of light on the activity of pure PAM

In order to investigate whether the activity of PAM was light dependent,
purified PAM (50 pg) was incubated with 1 mM of a-phenylalanine under light
illumination and non-illumination conditions, for a period of 1 hour or overnight.
Another pair of samples were set up with one exposed to UV light (254 nm) and
the other covered with aluminum or exposed to normal light. The amino acids

were derivatized after the incubation period and analyzed as described above.

4.5.2.6 Effect of sonication and light illumination on pure PAM
The effect of sonication was investigated by running 4 parallel reactions;
one in which a purified PAM-substrate mixture was sonicated for 5 min (1 min

pulse, 1 min intervals) on ice and another was incubated at room temperature

196

under total white light illumination, another was covered in aluminum foil and
incubated at room temperature and a control sample was incubated in ice for 10
min. The control sample kept on ice had no activity. The sonicated sample had
4% activity and the two samples kept at room temperature under dark and light

conditions respectively had 30 % activity.

4.5.2.7 Effect of type of radiation on PAM activity in E. coli.

E. coli BL21CodonPlus (DE3) RIPL colonies transformed with the pam
gene that was sub-cloned into vector pET14b97 were used to inoculate four 5 ml
of Luria Bertani broth (LB, 25 g/L) media containing 100 ug/uL of ampicillin and
50 ug/uL of chloromphenicol antibiotics. E. coli grown under non-illumination
were inoculated in tubes wrapped in aluminum foil to block light and incubated at
37 °C overnight. The 5 mL cultures were mixed, and 5 mL aliquots were
transferred into four 250 mL conical glass (PyrexTM) flasks containing 100 mL LB

media; two of the flasks were completely covered with aluminum foil and the
other two were left uncovered. The cultures were incubated at 37 °C until 00600
~ 0.6. The incubation temperature was reduced to 18 °C and the cultures were

induced with 1 mM isopropyl-B-D-thiogalactopyranoside (IPTG) to induce

expression of the pam gene. Each flask was exposed to similar light source, at
an intensity of 14 mW/cm2 from a separate Spot-Gro Sylvania light bulb
(maximum 770 lumens, 100 Color Rendering Index, 120 volts and 120 watts).

The lights were positioned outside of the shaker (50 cm) cabinet with a double-

walled glass door with 5 cm gap between panes. The distance between the lights

197

and the flasks was 137 cm. Two of the flasks (one covered with aluminum foil;
the other uncovered) were shielded with IR IF cut-off filters placed 2 cm in front
of the flasks and 135 cm away from the light bulbs. Under these conditions only

visible light between 295 to 850 nm with the maximum intensity at 546 nm is
irradiated. The intensity of light reaching the interior of the flasks was 27 mW/cm2

with a power of 0.97 mW, and the wavelength ranged from 300 to 750 nm with
the maximum intensity at 500 nm. The wavelengths range of light illuminating the
flasks was scanned and the intensities at the measured wavelengths were
compared to those provided by the manufacturer. Aliquots of cell cultures were
collected in duplicates (2 x 10 mL) from each flask at 6, 10, 12, 14, 16 and 18 h
after IPTG induction. To test the effects of blue light on the turnover of the PAM
in vivo, the above experiment was repeated except cut-off filter that reflects IR

and red light was used instead of only a filter that reflects IR.

4.5.2.7 Identifying phosphorylation in PAM using LC/MS/MS

Gel bands were subjected to in-gel tryptic digestion. The extracted
peptides were then automatically injected by a Waters nano Acquity Sample
Manager and loaded for 5 minutes onto a Waters Symmetry C18 peptide trap
(5pm, 180nm x 20mm) at 4 pL/min in 5% Acetonitrile/0.1% Formic Acid. The
bound peptides were then eluted onto a MICHROM Bioresources 0.1 x 150mm
column packed with 3p, 200A Magic C18AQ material over 35 minutes with a
gradient of 2% B to 35% B in 21 min, 90% B from 21-24 min and back to

constant 5% B at 24.1 min using a Waters nano Acquity UPLC (Buffer A = 99.9%

198

Water/0.1% Formic Acid, Buffer B = 99.9% Acetonitrile/0.1% Formic Acid) using
an initial flow rate of 800 nL/min, ramping to 1200 nL/min at 24 min and back to
800 nL/min at 35 min. Eluted peptides were sprayed into a Thermo Fisher LTO
Linear lon trap mass spectrometer outfitted with a MICHROM Bioresources
ADVANCE nano spray source. The top five ions in each survey scan were then
subjected to data-dependant zoom scans followed by low energy collision-
induced dissociation (ClD) and the resulting MS/MS spectra were converted to
peak lists using BioWorks Browser v 3.3.1 (Thermo Fisher) using the default LTO
instrument parameters. Peak lists were searched against all bacterial and green
plant protein sequences available in the NCBlnr database, downloaded on 11-
16-2008 from NCBI, using the Mascot searching algorithm, v2.2. The Mascot
output was then analyzed using Scaffold to probabilistically validate protein
identifications using the ProteinProphet computer algorithm. Assignments
validated above the Scaffold 95% confidence filter were considered true. Mascot
parameters for all databases were as follows: - allow up to 2 missed tryptic sites;
fixed modification of Carbamidomethyl Cysteine; - variable modification of
Oxidation of Methionine, Phosphorylation of Serine and Threonine; - peptide
tolerance of +/- 200ppm, - MS/MS tolerance of 0.8 Da; - peptide charge state

limited to +2/+3.

199

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216

CHAPTER 5

An Attempt to Decipher the Mechanism of Phenylalanine Aminomutase
(PAM) using 15N and 130 NMR

5.1 INTRODUCTION

The mechanism of the cofactor-less ammonia lyases and aminomutases
is believed to involve the nucleophilic attack of the substrate on the 3,5-dihydro-
5-methylidene-4H—imidazoI-4-one (MIO) of the enzyme."4 Two conflicting
theories have been postulated as to whether the phenyl ring or the amine group
of the substrate (or-phenylalanine) is the nucleophile and this has been debatable
for the past 45 years (Figure 5.1). The most recent contribution to this
mechanistic debate is based on the crystal structure of tyrosine aminomutase
from Streptomyces glabisporus (SgTAM)5'6 published by Christenson and co-
workers in which they show 2', 2'-difluorotyrosine as a pseudo-substrate bound to
the enzyme via the amine group of the substrate (Figure 5.2). The evidence in
support of the phenyl group of the substrate as the nucleophile is spearheaded
by Rétey and co-workers,7'20 based on theoretical calculations and broad
substrate specificities of phenylalanine ammonia lyase (PAL) and histidine
ammonia lyase (HAL). The trend of reactivity of the ring substituted

phenylalanine analogues appear to depend on the nucleophilicity of the phenyl

ring of the substrate.

217

 

 

 

H
\ COO
H
III
trans-cinnamate
PAL
I
K \ HN
H" N \ 4‘ \
QWN nix HIS
L
)H “J6

Figure 5.1 The two proposed mechanisms of PALs. On the left shows the aromatic ring of the
substrate attacking the MIO and on the right shows the amine group of the substrate attacking the

MIC.

218

 

 

Tyres Tyros
0
Y Gly70 D:\ Gil/70
o" , _ 9'
_ - 1,2 amine shift H
0201,. H ‘ A _ «\fi/Q
020‘ +
+ _
WNW—f- HZN O
__ o 574 0
MIO Nji‘x/K MIO Nle/K

 

 

 

Scheme 5.2 The 1, 2 shift of the amine group is initiated by the binding of the amine of the
substrate to the catalytic MIO cofactor. The mechanism is based on the crystal structure of
SgTAM.

The theoretical calculations by Rétey and coworkers are based on the
stability of the sigma complex formed between the phenyl ring of the substrate
and the MIO (Figure 5.1."). However, no comparison was made between the
energies of this transient state with that of the corresponding complex formed it
the amine was the nucleophile, which renders their data inconclusive. In support
of their conclusions and why the sigma complex would not collapse through a re-
aromatization through the loss of the 2'-proton, Rétey and coworkers present that
the aromatic ring is sandwiched between the MIO and phenylalanine399 (PAL R.
taluraides) which protects the proton by pi-pi interaction. The proton is positioned
perpendicular to the aromatic ring of and is hence ‘protected’ from de-
protonation. The same phenyl399 is believed to stabilize the highly unstable
carbocation generated on the ipso carbon. However no experiments have been

conducted to confirm these theoretical predictions. In this research we intend to

219

 

contribute to this unresolved mechanistic question, especially in consideration of
the mechanistic and functional differences between PALs and the target enzyme
phenylalanine aminomutase (PAM). Whereas the PALs just de-aminates, (Figure

5.3), PAM allows a partial rebounding of the amino group to the 6 position. The

aminomutases allows for the re-bounding of the NH3, possibly by restricting the

loss of the cinnamate intermediates from the active site, due to a narrow channel

21 -30

from the active site. Although PAM is functionally similar to SgTAM, it only

forms one isomer of the [3 acid whereas SgTAM has been observed to form both

the R and S [3 isomers at equilibrium, suggesting a slight mechanistic difference.

0 o
NH3
R/YLOH a R/VLOH
NHZ Ammonia Lyase

L-phenylalanine

\
m0

L-tyrosine

H
13%

K L-histidine

 

Scheme 5.3 General reaction of ammonia lyases. The aromatic amino acids are non-oxidatively
de-aminated to their corresponding 0, B-unsaturated acids.

220

5.1.2 Structural evidence for the amino group as nucleophile based on PAL
The first three-dimensional structure of phenylalanine ammonia lyase
(PAL) has been determined at 2.1 A resolution for PAL from Rhodosporidium

toruloides.31 ’32

The enzyme is structurally similar to the mechanistically related
histidine ammonia lyase (HAL), with PAL having an additional 160 residues
extending from the common fold.31 The catalysis of PAL is proposed to be

governed by the dipole moments of seven or helices oriented towards the active

site.31 The MIO cofactor resides atop the positive poles of three helices, to

putatively increase its electrophilicity.31 The helix dipoles appear to be fully
compatible with a model of phenylalanine docked in the active site of PAL in
which the amino group of substrate is bound to methylidene group of MIO as a
cationic ammonium. There are 12 highly conserved residues near the N- termini
of helices whose functions are; substrate recognition, MIO activation, product
release,18 proton donation, and/or polarizing the electrons in the phenyl ring of

substrate for increasing the acidity of the protons at C3. The dipole near the C-

terminus of one helix is proposed to increase the basicity of a histidine residue in

the active site.”35

The imidazolone is positioned to act as a general base that
abstracts the pro-S hydrogen from C3 of the substrate. A similar mechanism and
structure is proposed for HAL.31 Thus, the presence of the helix dipoles appears

incompatible with a proposed mechanism that proceeds through a carbocation

intermediate.

221

5.1.3 Use of NMR in mechanistic Investigation

Nuclear Magnet Resonance (NMR) can be used to qualitatively and
quantitatively identify compounds. The major challenge is its relatively low
sensitivity which may be improved by use of large amounts of substrates and/or
enzyme. In enzymatic assays further complications may arise from the low
solubility of some proteins. Even more challenging is the study of the mechanism
based on analyzing the intermediates which usually have a short lived half-life.
One strategy is to trap the intermediate by using a mutant of the enzyme which
will not allow the reaction to go to completion. For sufficiently slow reactions if the
enzyme can be sufficiently concentrated then the transition states can be
detected using NMR spectroscopy. Sensitivity can be further increased by either
isotopically labeling the amino acid residue which binds or react with the

substrate or by utilizing a labeled substrate. In this experiment we make use of

universally labeled a-phenylalanine (1309 and 15N-or-phenylalanine), to enhance

the signal-to-noise ratio. The change in the chemical shift of the 130 and 15N will

be monitored overtime and will be used to establish the nucleophile. If the amine
is the nucleophile it will covalently bond to the MIO and cause a relatively high
”N chemical shift. The chemical shift of the ”N of the amine is expected to shift
up field since the MIO is electron withdrawing. The aromatic carbons which are
three bands away from the N there are not expected to shift significantly,
although the C1, C2 and CS may change considerably. The binding of the amine

to the MIO will result in an E1cb elimination of the pro-(S)-hydrogen and the

amine as an NHz-MIO-Enz complex (Figure 5.4.Ill). An NH3-MlO-Enz complex is

222

 

then formed by protonation of the amine (Figure 5.4.IV) by an acidic tyrosine. A
1, 4 Michael addition would result in the NHg-MIO-Enz complex (V) binding to the
C3 position of a, B-unsaturated acid. The NHz-MIO bond then expected to break

to form the 6 acid and reconstituting the enzyme (Figure 5.4.Vl). Intermediate IV
is the cinnamic acid which is expected to have a much longer half life and can be

isolated.

223

H
CNHZ \jl-Enz W0 09

NI / VII

Flgure 5.4 The expected intermediates of the substrate during the course of the reaction based
on the assumption that the amine of the substrate is the nucleophile. The reaction is expected to
go through three intermediates II, III, IV, V and VI before the product VII is formed.

The change in the chemical shift if the phenyl group of the substrate is a
nucleophile will be a general downward shift of the aromatic carbons. The ipso
carbon (figure 54”) will be a signature transient carbocation with an expected
chemical shift above 200 pm, however it is expected to be too short lived to be

detected on the NMR time scale.

224

 

 

 

 

 

 

 

\ Tyr322
Tyr322 o
e \
O H
H O A O
NH2 H2N
3 J-Enz OjH’Enz
O ...—
W ' I N\ N— I"
NY - T
\ Tyr322
Tyr322 90
G
O NH2 0
O 1
\ OH
3 ‘ (NH OH E
3 O H’ "Z

 

 

 

Scheme 5.5 The schematic reaction mechanism proposed if the aromatic ring of the substrate is
the nucleophile. The ipso carbocation (II) is expected to be very short lived, although its detection
will provide valuable lnfonnation. The phenyl ring attacks the MIO to form the complex II. The
tyrosine residue de-protonates the B—H to form intermediate III. The free amine picks up the
proton from the tyrosine to form IV which is followed by the re-aromatization of the substrate and

the elimination of free NH3, which re-bounds to the C3 position to form the product.

225

 

 

5.2 RESULTS AND DISCUSSION

5.2.1 RESULTS
5.2.1.1 13C NMR spectrum of the substrate

In order to examine whether the aromatic ring or amine is the nucleophile,
the steady state turnover of [1309, 15N]-cr-phenylalanine by PAM was analyzed.
The 130 NMR of the substrate was collected prior to the addition of a catalytic
amount of PAM (Figure 5.6A). The only feature of the spectrum is that of
C9H11N02, with resonance at 41 ppm (m, PhCI-120(NH2)COOH), 57 ppm (dd,
PhCH20l-I(NH2)COOH), 126 ppm (m, aromatic), 183 ppm (d, C(NH2)COOH), 138

ppm (m, aromatic) (Figures 5.68, C and D respectively).

226

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

150 100 50
(B) b c
I fir I I I I I I I I I I f I I I I I I I I
55.0 50 0 45.0 40 0
(C) c + f
,9
d
I I I I I I I I I I I I I I I I I
140 135.0 130.0 125.0

 

 

 

 

 

 

 

 

Figure 5.6 13C-NMR of the uniformly 13C labeled substrate (A), expanded aliphatic region (B),
expanded aromatic region (C) and the carbonyl region (D), showing the C—C and/or C-N coupling.

227

5.2.1.2 Kinetic array of the PAM reaction monitored by NMR
The reaction was monitored by 130 NMR for a total period of 17 hrs with

spectrum acquisition every 30 min. The total acquisition was 6 min and a delay
time of 24 min. Altogether 33 spectra were collected. Selected spectra are shown
in figure 5.7 for the aliphatic region. The product started showing after 30 min of
incubation and is persistent throughout the time of the reaction. New peaks
appear at 41, 51 (aliphatic region), 135, 142 (aromatic region) and 178 for the
carbonyl. The aromatic peaks do not integrate with the aliphatic region
suggesting the presence of three compounds; 0 and B phenylalanine and

cinnamic acid which were confirmed by comparing with authentic standards.

228

 

 

 

 

 

 

 

 

 

2 ALL “A _ A .4. AL 4“. _._. .L..- .4 ... A _. ._
fl v—fi _ 7‘... v w w. w w W WW
A A“ h.“ A— ._._ A. ALL- L _. .A-L... AL“. ._. A u“ _.... A L.
v v——' fl -4 at w a 7— w
A- A“ J “L. A.“ .A- -L DAL AML. A “AAA... ... 4L— 4. L
v— rv Try fi fl...“ “*7 v w .— _f .4... ——
._... _. M 4A.... ALL. _. _. “4.. A .._A A... M
v "v +‘w—v w‘vv'v-v v— v" www— v—vr' w—v—W —vvvw—
.AMu44LA _._.__A_. .A‘ w A __4_ .... A. _._
w- v w w w. WWF—v— —-. ~— ~—
I ”I I IN“ 4 A L .. .. J.‘ .. _. _
———'~~w .___ .. v “WV—'7‘. v " vv WV _ fi_ 7‘
A........ W‘W‘ L“ ““fl “
w v. - w... w. w wwv, 7. W ' w w~~ we. .w WW.

 

 

4 A E _. ‘ - L ‘
w v u v - _.._ uyv v v

~A A: A A—I 4.441 “LA-fi- A AA AAA-A— ‘_—-—‘

v V v v w ‘1 v _' www.1— 'v v V—V—V'

A.. .-A.-‘ h ##A 1 . L A _‘-““_‘_ An-l AIL“ ‘4 A‘ ._. . AA:-
“v-rY—- wfiv—r—u “a... v. V... Vi. F... “xv v... ,w .— v- we v...—
I I I

I I I f I I I T I I I I I I I I I I I fl I I I r I I I I I l I I I

 

 

 

 

55 50 45 40 35 30 25 ppm

Figure 5.7 Array of 130 NMR spectra for the PAM reaction with isotopically enriched L-a-
phenylalanine. The lowest spectrum was acquired before adding the enzyme. One of every 3
scans is shown out of a total of 33 scans.

5.2.1.3 Identification of the products of the PAM reaction by 13C NMR

PAM (5 mg, 0.13 mM) was added to the solution of universally labeled a-
phenylalanine. After the first 30 min new signals were observed at chemical shifts
of 43 ppm (triplet, PhCH2(NH2)Cl-IgCOOH), 51 ppm (PhCI-l2(NH2), 122 ppm
(triplet, alkenic), 128-130 (m, aromatic), 135 ppm (q, aromatic), 137 ppm
(aromatic), 140 ppm (multiplet, aromatic) 142 ppm ( aromatic), 178 (d, C=O), 181
ppm ( d, C=O), and 182 (d, C=O) (Figure 5.88). Peaks at 41, 57, 126 and 182
are due to the unreacted a-phenylalanine (Figure 5.6). The appearance of new

signals is due to the formation of products and/or intermediates. One of the

229

products was confirmed as B-phenylalanine based on the NMR spectrum of an
aunthetic standard (Figure 5.93) which has matching resonances with the

biosynthetic product (Figure 5.9A) at 43 and 51.

230

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

150 100 50
(B)
I . . . . I . r . . I . . . .f. r . t I .
145 140.0 135.0 130.0 125.0
(C)
I I I FYI— I I I
180.0 175.0
I I I I I I I L.;—h I -‘I—‘ I. I -: I r I I I I I I I
55.0 50.0 45.0 40.0

 

 

 

Figure 5.8 130 NMR Spectra of the product after 16 h incubation with PAM. (A) full spectrum, (B)
an expanded aromatic region, (C) and expanded carbonyl region and (D) an expanded aliphatic
region.

231

 

 

(A)

 

 

 

(B)

 

 

l l l

T I I I I I I r I I r I I I I

150.0 100.0 50.0

 

 

 

Figure 5.9 Comparison of the 130 NMR spectrum of the biosynthetically made product (A) versus

the authentic B-phenylalanine (B).

5.2.1.4 Identification of intermediates

The PAM reaction (overnight) product was treated with 1 N HCI to

precipitate the 'protein and separate enzyme bound and free products and

intermediates. The residue was filtered through a cotton pad. The pH was re-

adjusted to 8.0 and the solution assayed with 13C NMR spectroscopy in 10%

232

D20 (Figure 5.10). The work-up product (Figure 5.10B) had no evidence of the a

and B-phenylalanine (Figure 5.10A).

 

r ' ' I ' ' ' ' I ' ' ' ' I
150.0 100.0 50.0

Figure 5.10 130 NMR of the PAM reaction product after work-up by precipitating the enzyme. No
a- and B-phenylalanine peaks are exhibited (c.f Figure 5.9A).

The work-up product was identified as trans-cinnamic acid and not the cis-
isomer as shown below (Figure 5.11) as confirmed by the spectra of authentic

trans-cinnamic acid (Figure 5110).

233

 

Cis-cinnamic acid

 

 

 

 

170 160 150 140 130 120 110
(B)
Biosynthetic product
I I I I I l l I I I l I I I T I I I I I I I I T I T I I l I l I T I I T I I T
170 160 150 140 130 120 110
(C)

trans-cinnamic acid

 

 

 

 

 

I I I I I r I I Ij I I I I I I I I I I I I I I I I I I I I I I I I I I rfi—r

170 160 150 140 130 120 110

Figure 5.11 130 NMR for Cis-cinnamic acid (A), biosynthetically synthesized product (B) and
trans-cinnamic acid (C). The chemical shifts of the biosynthesis product corresponds to the
authentic trans-cinnamic acid.

234

 

 

5.2.1.6 ‘5N MNR SPECTRA

The 15N NMR spectrum for the pure substrate without enzyme has a

single resonance at 34 ppm (Figure 5.12A). Within 15 minutes of adding enzyme
(0.13 mM) to substrate four new peaks appeared (Figure 5.128), at -10, 11, 36
and 80 ppm. After an hour only three peaks (11, 36 and 42 ppm) persist and two
of them are visible throughout the 16 hour incubation period (Figure 5.13). The
peak 80 ppm is characteristic of a nitrogen bound to an electron withdrawing
group such as an aromatic system (an N in aniline resonate at 60-70 ppm).
Although the intensity of the peak at 42 ppm remains constant throughout the
course of the reaction, there is an observed ‘negative NOE’ like feature on the 36
ppm peak which seems to decrease as the 11 ppm peak increases. The ‘NOE’

like behavior could be caused by the long longitudinal and transverse relaxation
times of the 15N nuclear (between 60 and 120 s) in small molecules. The

relaxation delay was set at 1.2 s in order to analyze enzyme-substrate complex
which have a short relaxation time. The frequent pulsing required for the kinetic

experiment may also have caused pulsing by greater than 90‘2 thus not allowing

the 15N in the substrate to sufficiently relax.

235

 

 

 

uklll" llmmmm-uwmmmummml lMAbM‘IMMMfllMthflfl M11141.)
1 20 1 00 80 60 40 20 0 -20 -40 ppm

Flgure 5.12 15N NMR for [1309, 15N]-phenylalanine with PAM. NMR spectrum taken (A) before

adding PAM and (B) 15 min after initiating the reaction by mixing enzyme with the substrate in an
NMR tube.

 

 

 

 

 

 

V W
'- ..‘.'. '7‘ w r w rwr'v... v v
‘AL-‘n L I “A A. AA #H-A‘
'7‘ - —4 I A - ’-
va A.‘ A ‘ _.‘- '1 'r
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1 20 1 00 80 60 40 20 0 -20 -40 ppm

Flgure 5.13 The 15N NMR spectrum of isotopically enriched substrate incubated with wild type
PAM over 16 hours. Initially 3 peaks are distinguishable at 11 ppm, 36 ppm and 44 ppm.

236

 

5.2.1.6 Relative kinetic rates of the substrate, trans-cinnamic acid and ,8-
phenylalanine reaction as measured by 13C NMR

The relative rates of formation of the products were measured by
comparing the integration of the peak areas of a and B phenylalanine and trans-
cinnamic acid over a 17 h period. The ratios of the concentrations are shown in
figure 5.14. The a-phenylalanine substrate decreases exponentially, whilst the B-
phenylalanine initially increases rapidly before it starts to exponentially decay as
it is converted to trans-cinnamic acid. The trans-cinnamic profile increases

constantly to an equilibrium and is most dominant product.

 

 

 

 

 

 

120
+%Alpha
100 —I—Beta
A %CA
80 ‘ ‘ ‘ . ‘ . ‘ . ‘ .

60

40

20

Relative concentration (1,6 and TCA

 

 

 

 

4‘7 f 7 T I I I I I f I I f I I I I I I I I I I I I I I'fi I I f
Nestseeeeeeeeeee
Time or scans

O
L

 

 

 

Figure 5.14 The plot of relative rates of formation of the substrate, B-phenylalanine and trans-
cinnamic acid. The mass balance is referenced to the substrate (5 mM). At equilibrium the a and
B phenylalanines have the same (~10 % each).

237

5.2.2 DISCUSSION

The equilibrium of the PAM reaction is 52% in favor of the product.”38

The stereochemistry of the product has been established and partial mechanism
of the reaction has been proposed.38 One of the mechanistic voids in the PAM

reaction and similar enzymes like HAL and PAL has been whether the aromatic

ring or the amino group of the substrate is the nucleophile which attacks the MIC.

5

A study carried out by Christenson on SgTAM, which is mechanistically similar

to PAM, concludes that the amine is the nucleophile. In order to confirm this we

used an NMR technique, in which we monitor the change in the chemical shifts of

the 130 and 15N nuclei during the course of the reaction. A universally isotopically

labeled substrate (U-13C9, 15N-phenylalanine) was incubated with PAM (Figure

5.3) and the formation of intermediates was monitored overnight by taking scans
every 30 min at 31 °C. The isotopically enriched substrate presents challenges in
that the chemical shifts around the aromatic region are not clearly resolved. The .
detection of intermediates required that they be in sufficient quantities to be
detected by NMR spectroscopy. Although the use of isotopically enriched
substrate gives distinct coupling pattern the disadvantage is the reduced signal to
noise ratio which makes the procedure less sensitive. The data presented in this
report seek to address the mechanistic questions of the PAM reaction as a

complimentary technique to crystal structure studies.

In PAL release of NH3 is faster than release of cinnamate, whereas in

9.19.39

HAL, the release of NH3 is slowest. The mechanism involving the aromatic

238

ring as the nucleophile is doubtful considering the structure of PAL which

31

Calabrese and co-workers proposed based on its crystal structure. If six

positively charged helices are pointing towards the aromatic ring of the substrate,
it makes the phenyl ring a poor nucleophile. Furthermore creation of an ipso
positive charge on the ring will be destabilizing and thermodynamically

unfavorable because of the expected charge to charge repulsion.

15N- NMR studies of the reaction of PAM with 15N labeled phenylalanine
demonstrated that the intermediates involve two species bearing 15M, intimating

that the amine could be the nucleophile (Figure 5.13). The 15N chemical shift

changes from 34 ppm in the free state to 36 ppm after 15 minutes of the reaction,
other resonances are observed at -11, 10 and 80 ppm. The peak at 80 ppm
signifies that the N is bound to an electron withdrawing group most likely an
aromatic group, which in this case could be the MIC. The 80 ppm and -10 ppm
peaks disappear after 30 minutes, as another peak at 44 ppm emerges Figure
5.138, lower spectrum). After 2 hours, only three peaks are visible at 44, 36 and
11 ppm. The 36 ppm peak gradually diminishes in size over a period of 12 hours,
a phenomena which is not clear, although it could arguably be due to nuclear

overhauser effect (NOE). The signal at 36 ppm is the unused substrate, the one

at 44 ppm is the B-product and the one at 11 ppm is likely to be NH3. Based on

‘ we can conclude that the

this data and the information from the literature“"3
amine is the nucleophile although more evidence is required to confirm the

identity of some of the compounds or intermediates formed during the reaction.

130 NMR Spectra
239

The ‘30 NMR collected in this work was not very useful in identifying the

intermediates and transition states as originally designed. However the results
were useful in confirming the product of the PAM reaction as B-phenylalanine
and trans-cinnamic acid (Figure 5.11). At equilibrium (17 h) the q and B-
phenylalanine were at 20% of the total mix with the trans-cinnamic acid making
up 80% of the mixture. Perhaps the use of mono-labeled substrate will assist in
increasing the sensitivity of the method by eliminating the carbon-carbon
coupling. Sensitivity could also be enhanced by using at least 3 mM of enzyme.
In summary the NMR studies and other studies reported elsewhere in this
report 4043 demonstrate that PAM is a multifunctional enzyme that can participate
in the following types of reactions; (1) reversible isomerization of o and B
phenylalanine; (2) synthesis of cinnamic acids from either a or [3 phenylalanine;
(3) synthesis of or and B phenylalanine from cinnamic acid and (4) trans-

amination reaction (Figure 5.15 ).

 

 

- - PAM = ' '
Acrylic acid NH3
, +
B-phenylalanlne ct-arylalanine

Figure 5.15 The role of PAM as an aminating enzyme. The a- and B-amino acids are formed
from their corresponding cinnamates.

240

OH HO
NH2 Cl

Cl
0

\
OH > OH
H O NHZ
HO TAM

Figure 5.16 The potential function of PAM as a transaminase based on the functionality of TAM a
mechanistically similar enzyme.

 

241

 

5.4 CONCLUSIONS AND RECOMMENDATIONS

The low enzyme concentration (only 0.13 mM) made it impossible to

generate enough intermediate species for detection by 130 NMR. The signal

shifts (46 ppm) observed for 15N strongly suggest the involvement of the amine
of the substrate in the reaction. However, this conclusion is based on elimination
rather than on direct evidence, since the 130 chemical shifts for the transient

states were below the detection limit. The formation of trans-cinnamic acid is very
significant and it constitutes 80 °/o of the product mix at equilibrium as confirmed
by both NMR and GC-MS analysis. The large pool of trans-cinnamic acid at
equilibrium makes PAM a multifunctional enzyme which acts both as an
ammonia lyase and aminomutase. This role makes PAM a versatile enzyme
compared to ammonia lyases which only participate in non-oxidative elimination
of ammonia from aromatic amino acids. Our results were confirmed by the
Janssen group which published their work during the course of our study. They
reported on the biosynthesis of both o- and 6- amino acids from trans-
cinnamates using PAM from T. chinensis an enzyme which is 99% identical to
our PAM from T. Canadensis. Our method provides a method of synthesizing
labeled a, [3 unsaturated phenylpropanoic acids and d- and B- aromatic amino
acids capitalizing on the broad specificity of PAM.

Given the low solubility of PAM, we recommend carrying out the NMR
experiment is whole cells. The major advantage of whole cell NMR is that no

protein purification is required and the enzyme is in much higher concentration

242

and more substrate can be used. The disadvantages are the possible

interferences from other cell components.

243

 

and more substrate can be used. The disadvantages are the possible

interferences from other cell components.

243

5.3 MATERIALS AND METHODS

5.3.1 Materials
The 15N and 130 labeled substrate (U-1309, 97-99%; 15N 97-99%

phenylalanine) was purchased from Cambridge Isotope Laboratories, Inc
(Andover, MA, USA). All other reagents were obtained from Sigma-Aldrich and

used without further purification.

5.3.2.1 Enzyme purification

Phenylalanine aminomutase (PAM) was purified to 95% homogeneity as
previously described. The protein concentration was calculated based on the
Bradford assay and the absorbance was measured on a DU 64 Coultier

Beckmann Ultraviolet/visible spectrophotometer at a wavelength of 595 and the
standard was constructed with a linear regression of B2 = 0.99. The purified PAM

was concentrated and buffer exchanged (50 mM, Phosphate, 300 mM NaCl and
250 mM lmidazole for 50 mM Phosphate, 5 °/o glycerol) by repeated ultra filtration

over an Amicon 50 kDa cutoff membrane.

5.3.2 NMFI

NMR spectra were recorded with a Varian 500 MHz spectrometer in 5 mm

NMR tubes at 30 °C. All 130 and 15N NMR experiments were performed in 50
mM Tris- or Phosphate buffer pH 8.0 with 10% 020 for NMR signal lock. The “*0

NMR spectra were collected using the 500 MHz NMR spectrometer operating at

frequency of 125.67 MHz with proton decoupling at 499.74 MHz. Spectra were

244

 

collected over a width of 33.9 kHz with 33-1024 transients. The pulse routine
used included a 1.5 s delay and a signal acquisition time of 15. 90° pulses were

used. The spectral width was 33.3 kHz and all spectra were acquired with 88 000

data points. 13C spectra were processed using 1 Hz line-broadening. All chemical
shifts (6) of 130 were referenced against tetramethylsilane (6 =0) or CDCI3 (77
ppm)-

All 15N-NMFI experiments were performed in 50 mM Tris-HCI buffer pH 8.0

or 50 mM Phosphate buffer, pH 8.0 with 10% (WV) 020 for NMR signal lock. The

Varian UnityPlus 500 operated at a frequency of 50.64 Hz for the 15N nuclei.
Inverse gated 1H decoupling was applied only during signal acquisition. Spectral

data were collected with a width of 10.0 kHz. All 15N spectra were collected with

32-1024 transients with a 1 3 pulse delay with signal acquisition times of 0.64 3.
All samples were acquired with 45° and 90° pulses. Exponential multiplication

and Fourier transform with 1, 5 or 10 Hz line broadening were utilized for data

processing of 15N spectra. All chemical shifts (6) of 15N were referenced to
110.0ppm for external 15N-labelled formamide, but reported against liquid NH3 (6

= 0 Hz). 15N spectra were collected over a 480 ppm range to assure observation

of all N species.

245

 

5.3.3 Preparation of Cis-CA crystals

Approximately 1.5 g of trans-CA was dissolved in 96% ethanol and
irradiated under ultraviolet-light for overnight. The mixture of E (trans-isoform)
and Z (cis-isoform) isomers of cinnamic acid was re-dissolved in ethanol and
separated on HPLC. Crystals of cis-CA were precipitated out of a cis-CA solution

under vacuum. 1H nuclear magnetic resonance (NMR) was used to authenticate
the structure of cis-CA and trans-CA as previously described.“59 Further

qualitative assay were confirmed using GC-MS.

5.3.4 High performance liquid chromatography.

The high performance liquid chromatography (HPLC) used to purify cis-
CA consists of Waters 600 controller, 600 pump, with a UV/visible detector.
Separation of cis-CA from trans-cinnamic acid was achieved on a normal phase
Astec ChirobioticTM (25 cm x 4.6 mm, 5 (M). The mobile phase was a mixture of
acetonitrile (buffer A) and acetic acid (buffer B). The ratio of buffer A to B was set
to 3 I 7 at the initial stage and changed gradually to 4 I 6 by 8 min and finally to
4.5 5.5 by 30 min. The flow rate was set at 1.0 mL/min. The wavelength of UV-
light detector was set at 261 nm because of the Cis-CA absorption peaks at this
wavelength. The retention time of authentic Cis-CA standard on HPLC was

determined to be 2.50 :l: 0.25 min.

246

 

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