Active Site Engineering of ω-Transaminase for Production of ...

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(Enzymology and Protein Engineering) 1

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Active Site Engineering of ω-Transaminase for Production of Unnatural 3

Amino Acids Carrying a Side Chain Bulkier Than an Ethyl Substituent 4

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Sang-Woo Han, Eul-Soo Park, Joo-Young Dong and Jong-Shik Shin* 7

Department of Biotechnology, Yonsei University, Seoul 120-749, South Korea 8

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*To whom correspondence should be addressed. 11

Engineering Building 2 (Rm 507), Yonsei University, Shinchon-Dong 134, Seodaemun-Gu, 12

Seoul 120-749, South Korea (E-mail: [email protected], Tel: (+82) 2-2123-5884, Fax: 13

(+82) 2-362-7265) 14

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Running Title: Engineering substrate specificity of ω-transaminase18

AEM Accepted Manuscript Posted Online 31 July 2015Appl. Environ. Microbiol. doi:10.1128/AEM.01533-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 19

ω-Transaminase (ω-TA) is a promising enzyme for production of unnatural amino acids from 20

keto acids using cheap amino donors such as isopropylamine. The small substrate binding 21

pocket of most ω-TAs permits entry of substituents no larger than an ethyl group, which 22

presents a significant challenge to the preparation of structurally diverse unnatural amino acids. 23

Here we report engineering of an (S)-selective ω-TA from Ochrobactrum anthropi (OATA) to 24

reduce the steric constraint and thereby allow the small pocket to readily accept bulky 25

substituents. Based on a docking model using L-alanine as a ligand, nine active site residues 26

were selected for alanine scanning mutagenesis. Among the resulting variants, a L57A variant 27

showed dramatic activity improvements for α-keto acids and α-amino acids carrying up to a n-28

butyl substituent (e.g. 48 and 56-fold activity increases for 2-oxopentanoic acid and L-norvaline, 29

respectively). A L57G mutation also relieved the steric constraint but much less than the L57A 30

mutation did. In contrast, a L57V substitution failed to induce the activity improvements for 31

bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in 32

a large pocket, induces an altered binding orientation of an α-carboxyl group and thereby 33

provides more room to the small pocket. Synthetic utility of the L57A variant was demonstrated 34

by carrying out production of optically pure L and D-norvaline (i.e. ee > 99 %) by asymmetric 35

amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively. 36

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INTRODUCTION 42

Unnatural amino acids are widely used as essential chiral building blocks for diverse 43

pharmaceutical drugs, agrochemicals and chiral ligands (1-3). In contrast to natural amino acids, 44

a fermentative method is not yet commercially available for production of the unnatural amino 45

acids (4, 5). This has driven development of various biocatalytic approaches to afford scalable 46

processes for preparing enantiopure unnatural amino acids, which includes kinetic resolution of 47

racemic amino acids using acylase (6, 7), amidase (8, 9), hydantoinase (10, 11) and amino acid 48

oxidase (12, 13), and asymmetric reductive amination of keto acids using dehydrogenase (14, 49

15) and transaminase (16, 17). The asymmetric amination is usually favoured over the kinetic 50

resolution because the former affords 100 % yield without racemization of an unwanted 51

enantiomer. 52

In contrast to a mandatory requirement of supply and regeneration of an expensive external 53

cofactor for the dehydrogenase reactions, transaminase catalyzes transfer of an amino group 54

from an amino donor to an acceptor using pyridoxal 5′-phosphate (PLP) as a prosthetic group 55

(18). Nevertheless, industrial implementation of the transaminase-mediated synthesis of 56

unnatural amino acids has lagged behind because of low equilibrium constants, usually close to 57

unity, for the reactions between keto acids and amino acids. However, recent studies have 58

demonstrated that reductive amination of keto acids can be driven to completion without the 59

thermodynamic limitation by employing cheap amines as a cosubstrate when the transfer of an 60

amino group can be mediated by ω-transaminase (ω-TA) that utilizes primary amines as an 61

amino donor (19-21). For example, the equilibrium constant for amination of pyruvic acid by 62

isopropylamine was reported to be 67 (22). Therefore, use of 1.5 molar equivalent of 63

isopropylamine relative to pyruvic acid affords 97 % theoretical conversion of pyruvic acid to L 64


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or D-alanine, depending on the stereoselectivity of ω-TA. Moreover, acetone (i.e. the 65

deamination product of isopropylamine) is highly volatile and the resulting equilibrium shift by 66

facile evaporation can drive even thermodynamically unfavorable reactions to completion as 67

demonstrated elsewhere with reductive amination of ketones (23-25). Another advantage of 68

using isopropylamine as an amino donor is that most ω-TAs show low activities for acetone 69

(26), which minimizes enzyme inhibition by the ketone product. Taken together, ω-TA-70

catalyzed asymmetric amination of keto acids using isopropylamine is a promising strategy for 71

scalable production of unnatural amino acids. 72

One crucial bottleneck for synthesizing diverse unnatural amino acids using ω-TAs is very 73

narrow substrate specificity of the enzyme toward keto acids. It is generally accepted that both 74

(R) and (S)-selective ω-TAs possess two substrate-binding pockets consisting of a large pocket 75

capable of dual recognition of hydrophobic and carboxyl groups and a small pocket that can 76

accept substituents no larger than an ethyl group (27, 28). The canonical structural features of 77

the active site of ω-TAs render a carboxyl group and a side chain of keto acid substrates bound 78

to the large and small pockets, respectively. This leads all known ω-TAs to accept a limited 79

range of α-keto acids such as glyoxylic acid, pyruvic acid and 2-oxobutyric acid. The only 80

exception to the canonical substrate specificity to date is an (S)-selective ω-TA from 81

Paracoccus denitrificans that can accommodate up to a n-butyl substituent of α-keto acid in the 82

small pocket (29). We demonstrated that a single point mutation in the small pocket (i.e. 83

V153A) endowed the ω-TA with a substantial activity toward even 2-oxooctanoic acid carrying 84

a n-hexyl side chain (29). Despite the non-canonical steric constraint of the ω-TA from P. 85

denitrificans, a low enzyme activity for isopropylamine renders a synthetic potential of the ω-86

TA less optimal for practical amination of the bulky keto acids (21). This led us to set out 87


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engineering of substrate specificity of the canonical ω-TAs showing a high activity for 88

isopropylamine. To this end, we chose an (S)-selective ω-TA from Ochrobactrum anthropi 89

(OATA) which showed 43 % activity for isopropylamine relative to that for (S)-α-90

methylbenzylamine ((S)-α-MBA) (21). 91

To date, there have been three reports dealing with engineering of the canonical steric 92

constraint in the small pocket of ω-TAs. In the first example, an (R)-selective ω-TA from 93

Arthrobacter sp. was successfully engineered to create a variant that can accept bulky arylalkyl 94

ketones as an amino acceptor (23). In our previous study, substrate specificity of the engineered 95

variant, harboring 27 amino acid substitutions, was examined using structurally diverse α-keto 96

acids (21). In spite of the remarkable activity improvements for bulky arylalkyl ketones, the 97

canonical steric constraint of a parental enzyme toward α-keto acids was not significantly 98

altered in the engineered variant, e.g. only 2 % reactivity of 2-oxopentanoic acid relative to 99

pyruvic acid. This result suggests that engineering of the steric constraint should be guided in 100

the context of a substrate type, especially substituents bound to the large pocket (i.e. 101

hydrophobic vs carboxyl). The second example was carried out with an (S)-selective ω-TA 102

from Vibrio fluvialis to improve an activity toward a β-keto ester (i.e. (R)-ethyl 5-methyl 3-103

oxooctanoate) (30). The resulting variant, carrying eight amino acid substitutions, showed a 60-104

fold activity increase for the target β-keto ester but led to a complete activity loss for keto acids. 105

The third example employed the same ω-TA as the one used in the second example and focused 106

on improving activities for arylalkyl ketones. To the best of our knowledge, active site 107

engineering of a ω-TA to relieve the canonical steric constraint for α-keto acids and α-amino 108

acids has not yet reported. 109


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In this study, we aimed at creating a variant of OATA capable of accommodating bulky α-110

keto acids. To this end, we performed substrate docking simulations to select key active site 111

residues and carried out alanine scanning mutagenesis to identify a hot spot responsible for the 112

narrow substrate specificity for α-keto acids. The resulting variant carrying only a single point 113

mutation in the large pocket showed desirable relaxation of the canonical steric constraint and 114

afforded efficient stereoselective amination of bulky α-keto acids using isopropylamine as an 115

amino donor. 116

MATERIALS AND METHODS 117

Chemicals 118

Pyruvic acid was obtained from Kanto Chemical Co. (Tokyo, Japan). Isopropylamine was 119

purchased from Junsei Chemical Co. (Tokyo, Japan). L-Alanine was purchased from Acros 120

Organics Co. (Geel, Belgium). All other chemicals were purchased from Sigma Aldrich Co. (St. 121

Louis, MO). Materials used for preparation of culture media including yeast extract, tryptone 122

and agar were purchased from BD Biosciences (Franklin Lakes, NJ). 123

Site-directed mutagenesis of ω-TA 124

Single point mutations of OATA were carried out using a QuikChange Lightning site-directed 125

mutagenesis kit (Agilent Technologies Co., Santa Clara, CA) according to an instruction 126

manual. The template used for the mutagenesis PCR was pET28-OATA which was previously 127

constructed (31). Mutagenesis primers were designed by a primer design program 128

(http://www.agilent.com). The primer sequences are provided in Table 1. Intended mutagenesis 129

was confirmed by DNA sequencing. 130


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Expression and purification of ω-TAs 131

Overexpression of His6-tagged ω-TAs was carried out as described previously with minor 132

modifications (32). Escherichia coli BL21(DE3) cells carrying the expression vectors (i.e. 133

pET28a(+) harboring the ω-TA gene) were cultivated in LB medium (typically 1 L) containing 134

50 μg/mL kanamycin. Protein expression was induced by IPTG at 0.4 OD600 and the cells were 135

allowed to grow for 10 h. The culture broth was centrifuged and the resulting cell suspension 136

was subjected to ultrasonic disruption. Protein purification was carried out as described 137

previously (32). Protein purity was confirmed by SDS-PAGE (Fig. S1). Molar concentrations 138

of the purified ω-TAs were determined by measuring UV absorbance at 280 nm. 139

Enzyme assay 140

All enzyme assays were carried out at 37 °C and pH 7 (50 mM phosphate buffer). Unless 141

otherwise specified, reaction conditions for the activity assay were 10 mM (S)-α-MBA and 10 142

mM pyruvic acid. Typical reaction volume was 50 μL. The enzyme reaction was stopped after 143

10 min by adding 300 µL acetonitrile. Acetophenone produced from the reactions was analyzed 144

by HPLC. For initial rate measurements, reaction conversions were limited less than 10 %. 145

Substrate specificity 146

To examine substrate specificity of the OATA variants, initial rate measurements (i.e. < 10 % 147

conversion) were independently triplicated. Reaction conditions to examine substrate 148

specificity for amino acceptors were 20 mM α-keto acid and 20 mM (S)-α-MBA in 50 mM 149

phosphate buffer (pH 7). Acetophenone produced was analyzed by HPLC. To measure 150

activities for α-amino acids, 20 mM amino donor and 20 mM propanal were used as substrates 151

and the α-keto acids produced were analyzed by HPLC. To measure amino donor activities for 152


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isopropylamine, reaction conditions were 20 mM isopropylamine and 20 mM pyruvic acid. L-153

alanine was analyzed by chiral HPLC after derivatization with a Marfey’s reagent (33, 34). 154

Molecular modeling 155

Using four X-ray structures of (S)-selective ω-TAs as templates, a homology model of OATA 156

was constructed by the Modeler module (version 9.8) of the Discovery Studio package (version 157

3.5.0, BIOVIA, San Diego, CA). X-ray structures used as templates were ω-TAs from P. 158

denitrificans (PDB ID: 4GRX) (35), Chromobacterium violaceum (4A6T) (36), Mesorhizobium 159

loti (3GJU) (37) and Rhodobacter sphaeroides (3I5T) (37). The outward-pointing arginine of 160

4GRX was set to be conserved in the homology model, resulting in a dimeric structure of 161

OATA in which each subunit has a different conformation of the active-site arginine. To 162

construct a holoenzyme structure, the PLP moiety was copied from 4A6T. Only 0.4 % non-163

glycine residues were found to lie in the disallowed region by a Ramachandran phi-psi analysis. 164

The active site models of the L57A and L57G variants were built by an amino acid 165

substitution and then energy minimization (2,000 steps; dielectric constant = 4) of the mutation 166

site, the internal aldimine and the neighboring residues within 3 Å from L57 (i.e. S55, G56, 167

L57, W58, S59, F82, H84 and T324) until the RMS gradient reached 0.1 kcal/mol/Å. 168

Docking simulations with L-alanine and L-norleucine as ligands were accomplished using 169

the CDOCKER module under a default setting within the active site defined by the Binding-170

Site module. The most stable docking pose was chosen as a docking model. 171

Kinetic analysis 172

A pseudo-one-substrate kinetic model was used to obtain apparent kinetic parameters for 173

pyruvic acid and 2-oxohexanoic acid as described previously (38). Concentration range of 174


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pyruvic acid for initial rate measurements was 0.5 - 5 mM. Concentration ranges of 2-175

oxohexanoic acid were 7 - 200 mM and 1 - 30 mM for the wild-type and the L57A variant, 176

respectively. Concentration of the cosubstrate (i.e. (S)-α-MBA) was fixed at 20 mM. 177

Acetophenone produced was analyzed by HPLC to measure the initial rates. The initial rate 178

data were fitted to a Michaelis-Menten equation, and the KM and kcat values were calculated 179

from the slopes and y-intercepts of the double-reciprocal plots. 180

Measurement of enzyme stability 181

To examine the effect of the L57A mutation on enzyme stability, 10 μM enzyme was incubated 182

in 50 mM potassium phosphate buffer (pH 7) at 37 oC. Aliquots of the enzyme solution were 183

sampled at predetermined incubation times and were mixed with reaction buffer containing 10 184

mM pyruvic acid and 10 mM (S)-α-MBA. Initial reaction rates were measured by analyzing 185

acetophenone produced. Inactivation constants were obtained by curve fitting of the residual 186

activity data to a single exponential function. 187

Enzyme reactions to produce unnatural amino acids 188

The reaction volume for the enzyme reactions was 1 mL and the reaction mixture in 50 mM 189

potassium phosphate buffer (pH 7) was incubated at 37 °C. Reaction conditions for asymmetric 190

synthesis of L-norvaline were 50 mM 2-oxopentanoic acid, 100 mM isopropylamine and 40 191

μM ω-TA. Synthesis of L-norleucine was carried out under the reaction conditions of 100 mM 192

2-oxohexanoic acid, 200 mM isopropylamine, 0.1 mM PLP and 200 μM ω-TA. Kinetic 193

resolution of racemic norvaline was performed at 50 mM rac-norvaline, 50 mM glyoxylic acid 194

and 40 μM ω-TA. Aliquots of the reaction mixture (50 μL) were taken at predetermined 195

reaction times and mixed with 10 μL HCl solution (5 N) to stop the reaction. The reaction 196


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mixtures were subjected to HPLC analysis for measurement of conversion and enantiomeric 197

excess. 198

HPLC analysis 199

All the HPLC analyses were performed on a Waters HPLC system (Milford, MA). Analysis of 200

acetophenone was performed using a Sunfire C18 column (Waters Co.) with isocratic elution of 201

60 % methanol/40 % water/0.1 % trifluoroacetic acid at 1 mL/min. UV detection was done at 202

254 nm. Quantitative chiral analyses of alanine, norvaline and norleucine were carried out using 203

a Crownpak CR(-) column (Daicel Co., Japan) or a Sunfire C18 column after chiral 204

derivatization with a Marfey’s reagent (33, 34). Κeto acids were analyzed using an Aminex 205

HPX-87H column (Bio-Rad, Hercules, CA) with isocratic elution of 5 mM H2SO4 solution at 206

0.5 mL/min. Column oven temperature was set to 40 oC and UV detection was done at 210 nm. 207

RESULTS AND DISCUSSION 208

Substrate docking 209

We set out to construct a homology model of OATA to identify key active site residues 210

responsible for rejecting entry of bulky substituents in the small pocket. X-ray structures used 211

as templates for the homology modeling were ω-TAs from P. denitrificans (PDB ID: 4GRX, 212

sequence identity: 41 %) (35), C. violaceum (4A6T, 41 %) (36), M. loti (3GJU, 43 %) (37) and 213

R. sphaeroides (3I5T, 34 %) (37). Sequence alignment of OATA against the template ω-TAs is 214

shown in Fig. S2. The four template ω-TAs adopt a homodimeric structure where both active 215

site arginines assume an inward conformation (i.e. pointing toward the large pocket), except 216

4GRX where one subunit harbors an outward arginine (i.e. pointing toward a solvent side). It is 217


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known that the active site arginine, responsible for recognition of a carboxylate of an incoming 218

substrate, undergoes a gross movement and assumes the outward conformation when the large 219

pocket is taken up by a hydrophobic substituent (39). 220

We performed substrate docking simulations using the subunit structure where the active 221

site arginine (i.e. R417) adopts the inward conformation (Fig. 1). For the docking simulation, 222

we used L-alanine, instead of pyruvic acid, as a ligand because all the active site lysines in the 223

template ω-TAs form a Schiff base with PLP (i.e. an internal aldimine capable of deamination 224

of an amino donor) and thereby the resulting homology model adopts the internal aldimine 225

structure. It is notable that binding of L-alanine is coordinated by a hydrogen bond between the 226

amino group of the substrate and the phenolic oxygen of the PLP moiety as well as multiple 227

hydrogen bonds between the α-carboxylate and the active site arginine. Compared to the active 228

site structure where R417 adopts an outward conformation, the large pocket becomes 229

contracted owing to the inward conformation of R417 to recognize the carboxyl group. The 230

small pocket accommodating the methyl group of L-alanine is wrapped by side chains of the 231

five active site residues (i.e. Y20, F86, Y151, V154 and T324), a phosphate group of the PLP 232

and a backbone chain ranging from G322 to T324. The Connolly surface of the active site 233

clearly explains why the small pocket can accept only up to an ethyl substituent. 234

235

Alanine scanning mutagenesis of the active site residues 236

The docking model indentified thirteen active site residues that participate in the formation of 237

the active site surface and lie within a 7 Å distance from the bound L-alanine (i.e. Y20, L57, 238

W58, F86, Y151, V154, A230, I261, K287, G322, F323, T324 and R417). We carried out 239

alanine substitution of the active site residues one by one to examine size reduction of which 240


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residue led to generation of more room required for accepting bulky substituents in the small 241

pocket. A230 and G322 were excluded from the mutagenesis because alanine substitution of the 242

two residues could not generate more room in the active site. K287 and R417 were also 243

excluded because these are essential residues responsible for catalytic turnover and recognition 244

of a carboxyl group, respectively. Therefore, nine active site residues were subjected to the 245

alanine scanning mutagenesis and the enzyme activities of the resulting variants toward 2-246

oxopentanoic acid were measured (Fig. 2). Among the nine variants, a L57A variant showed an 247

exceptionally improved activity for 2-oxopentanoic acid (i.e. a 48-fold activity increase relative 248

to the wild-type enzyme). This was an unexpected result because the L57 residue participates in 249

the large pocket. Besides L57A, V154A was the only alanine substitution that allowed activity 250

improvement for 2-oxopentanoic acid (i.e. a 3-fold increase). This result is in line with our 251

previous observation where alanine substitution at the same position of the ω-TA from P. 252

denitrificans achieved excavation of the small pocket (29). 253

Site-directed mutagenesis of L57 254

We presumed that the remarkable activity improvement for 2-oxopentanoic acid by the L57A 255

mutation would result from altered binding of a carboxyl group owing to generation of more 256

room in the large pocket by the size reduction of L57. To examine whether a higher activity 257

improvement could be induced by substitution of L57 with small amino acids other than alanine, 258

we prepared L57V and L57G variants by site-directed mutagenesis. Enzyme activities for 259

various α-keto acids were measured in comparison with the wild-type enzyme and the L57A 260

variant (Table 2). The wild-type OATA showed negligible activities (i.e. < 1 % relative to 261

pyruvic acid) for bulky α-keto acids carrying a side chain larger than an ethyl group. The L57V 262

mutation did not induce any activity improvement for the bulky α-keto acids but caused drastic 263


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activity losses toward native substrates (i.e. from glyoxylic acid to 2-oxobutanoic acid). In 264

contrast, the L57A variant showed activity improvements for α-keto acids carrying side chains 265

larger than a methyl group (i.e. 2, 48 and 39-fold activity increases for 2-oxobutyric acid, 2-266

oxopentanoic acid and 2-oxohexanoic acid, respectively). The activity improvements for bulky 267

α-keto acids were so remarkable that the L57A variant exhibited a substantial activity even for 268

2-oxooctanoic acid (i.e. 2 % relative to pyruvic acid). The L57G substitution also induced the 269

activity improvements for bulky α-keto acids but to a much lesser degree than those observed 270

with the L57A substitution. Despite the activity improvements toward α-keto acids carrying 271

bulky linear alkyl groups, neither L57A nor L57G variant displayed substantial activities for 272

branched-chain α-keto acids, including 3-methyl-2-oxobutyric acid, 3-methyl-2-oxopentanoic 273

acid, 4-methyl-2-oxopentanoic acid and trimethylpyruvic acid (data not shown). 274

As shown in Table 3, the relaxed steric constraint for α-keto acids was also observed for 275

α-amino acids (i.e. 56 and 5-fold activity improvements of the L57A and L57G variants, 276

respectively, for L-norvaline). Despite the nondetectable activity of the wild-type OATA for L-277

norleucine, the relaxed steric constraint allowed the L57A and L57G variants to show 278

substantial activities for L-norleucine. The L57V mutation led to drastic activity decreases for 279

α-amino acids as also observed with α-keto acids. Interestingly, all the OATA variants in 280

addition to the wild-type showed very low activities for glycine despite the high activities for 281

the cognate keto acid. These extremely opposite activities for glycine and glyoxylic acid seem 282

to be ascribable to the thermodynamically unfavorable conversion of glycine to its keto acid 283

(40). As observed with branched-chain α-keto acids, both L57A and L57G variants showed 284

nondetectable activities for α-amino acids carrying branched-chain substituents, including L-285

valine, L-leucine, L-isoleucine and L-tert-leucine (data not shown). 286


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The synthetic utility of the L57A variant for asymmetric amination of bulky keto acids is 287

contingent upon high activity for isopropylamine as well as no loss in the parental 288

stereoselectivity. We measured amino donor reactivity of isopropylamine with the L57 variants 289

(Fig. 3). In contrast to L57V and L57G variants, the L57A variant conserved 80 % of a parental 290

activity for isopropylamine. In addition to the wild-type OATA, all the L57 variants showed 291

nondetectable formation of D-alanine during the transamination between isopropylamine and 292

pyruvic acid (i.e. ee of L-alanine produced > 99.9 %). This result indicates that the three amino 293

acid substitutions of L57 do not affect the stringent stereoselectivity of the parental enzyme. 294

295

Structural modeling of the L57A variant 296

To provide a structural insight into how the L57A mutation relieved the steric constraint in the 297

small pocket, we performed docking simulation of the L57A variant using L-alanine as a ligand 298

(Fig. 4A). Compared to the docking pose of L-alanine in the wild-type active site, the L57A 299

mutation allowed docking of L-alanine slightly away from the small pocket with 0.71 and 0.73 300

Å translocations of the C-α and C-β of the substrate, respectively, to the large pocket. These 301

translocations are attributed to a 1.35 Å movement of the α-carboxyl carbon to the W58 residue 302

owing to the room created by the L57A substitution. The altered binding orientation of the α-303

carboxylate in the large pocket permits an additional hydrogen bond formation with the indole 304

group of W58, which led to a concurrent loss in one of the pre-existing hydrogen bonds with 305

R417. 306

The altered binding of the α-carboxylate in the active site of the L57A variant led to the 307

substrate docking more close to the large pocket, which provides more effective room to the 308

small pocket even without any structural change in the small pocket. In the substrate specificity 309


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experiments, we could test neither α-keto acid nor α-amino acid substrates carrying a n-pentyl 310

side chain because these substrates were not commercially available. To examine how large 311

substituent the extended small pocket can accommodate, we carried out docking simulation 312

using L-norleucine as a ligand (Fig. 4B). The docking model is consistent with the significant 313

enzyme activity for L-norleucine and predicts that the small pocket cannot accept a n-pentyl 314

group. The n-butyl group of the bound L-norleucine does not assume a staggered conformation 315

due to a steric interference with the small pocket residues, resulting in an intramolecular steric 316

strain of the bound substrate. This could explain the 87 % activity loss for L-norleucine 317

compared to L-alanine. 318

Contrary to the expectation, the L57G variant did not show higher activities toward 319

bulky substrates than the L57A variant did. To examine how much the L57G mutation allowed 320

the altered binding of the α-carboxyl group to the large pocket, we performed docking 321

simulation of L-norleucine in the active site of the L57G variant (Fig. S3). The docking pose of 322

L-norleucine in the L57G variant turned out very close to that generated with the L57A variant, 323

indicating that creation of a bigger room in the large pocket by the L57G mutation did not elicit 324

larger translocation of the α-carboxyl group toward the large pocket. This is in agreement with 325

the docking model of L-norleucine in the L57A variant where the α-carboxyl group is already 326

in a close contact with W58 and thereby further translocation of the α-carboxyl group is 327

prohibited because of a steric clash against W58 (Fig. 4B). It is presumable that the lower 328

activities of the L57G variant for bulky substrates, compared to the L57A variant, result from 329

increased conformation flexibility of the backbone chain which might be deleterious to a 330

catalytic step after formation of a Michaelis complex. This is supported by the drastic activity 331

losses of the L57G variant even for the native substrates such as glyoxylic acid, pyruvic acid 332


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and L-alanine. Note that two consecutive glycine residues happen to be created immediately 333

before W58 in the L57G variant because the 56th residue is glycine (Fig. S2). 334

Kinetic analysis of the L57A variant 335

To provide mechanistic understanding of how the L57A mutation improves activities for bulky 336

substrates, we compared kinetic parameters of the wild-type and the L57A variant for pyruvic 337

acid and 2-oxohexanoic acid (Table 4). Compared with the wild-type enzyme, the L57A variant 338

showed a 50 % reduction in the specificity constant (i.e. kcat/KM) for pyruvic acid. This activity 339

loss for the native substrate is caused by a 2.8-fold decrease in the binding affinity although the 340

L57A mutation induced a 1.4-fold increase in the turnover number. The weakened binding to 341

pyruvic acid by the L57A mutation seems to result from the loss of a native hydrogen bond 342

between R417 and the α-carboxylate of pyruvic acid (Fig. 1 and Fig. 4B). 343

As the side chain of the α-keto acid substrate extends from a methyl to a n-butyl group 344

(i.e. from pyruvic acid to 2-oxohexanoic acid), the wild-type enzyme showed a 290- and 110-345

fold decreases in the binding affinity and the turnover number, respectively. The deterioration 346

in both binding and catalytic steps renders the wild-type enzyme near inactive toward 2-347

oxohexanoic acid. The L57A mutation turned out to promote both steps (i.e. 6- and 48-fold 348

increases in the binding affinity and the catalytic turnover, respectively), leading to a 290-fold 349

increase in the specificity constant for 2-oxohexanoic acid. It is notable that the fold-change in 350

kcat is even larger than that in KM, indicating that the L57A-induced alleviation of the steric 351

interference in the small pocket benefits the catalytic step much more than the binding step. 352

This result is in accordance with the structural model of a quinonoid intermediate (i.e. the most 353

unstable reaction intermediate) where formation of a covalent linkage between substrate and 354

PLP rearranges the substrate moiety close to the PLP side and thereby results in stronger steric 355


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interference of a bulky substituent in the reaction intermediate than that in the Michaelis 356

complex (38). 357

Synthetic utility of the L57A variant 358

For practical applicability of enzyme variants engineered to attain a desirable functionality, it is 359

essential for the amino acid substitution to disturb the protein stability in a minimal way. We 360

performed time-course monitoring of the enzyme activities under the incubation conditions of 361

50 mM phosphate buffer (pH 7.0) and 37 °C which were the same as those used in the reaction 362

mixture for a preparative purpose (Fig. 5). Intriguingly, we found that the L57A mutation 363

remarkably increased enzyme stability. Inactivation constants obtained from curve fitting of the 364

residual activity data to a single exponential function were 1.1×10-2 h-1 (r2 = 0.97, half life = 62 365

h) and 8.1×10-4 h-1 (r2 = 0.89, 860 h) for the wild-type and the L57A variant, respectively. The 366

striking stability improvement renders the L57A variant highly promising for industrial 367

applications. 368

To examine how the L57A mutation improves the catalytic potential of OATA for 369

production of bulky unnatural amino acids, we carried out asymmetric synthesis of L-norvaline 370

which is a key intermediate of Perindopril (i.e. an ACE inhibitor) (41) and a potential inhibitor 371

of arginase (42). As expected, the L57A variant afforded much faster synthesis of L-norvaline 372

from 50 mM 2-oxopentanoic acid and 100 mM isopropylamine than its parental enzyme did at 373

the same reaction conditions (Fig. 6A). Conversion reached 99.3 % at 2 h with > 99.9 % ee of 374

the resulting L-norvaline using the L57A variant, whereas the wild-type OATA permitted only 375

22.6 % conversion at 2 h. As shown in Fig. 6B, we also performed asymmetric synthesis of L-376

norleucine from 100 mM 2-oxohexanoic acid and 200 mM isopropylamine using a 5-fold 377

higher enzyme concentration than that used in Fig. 6A. The L57A variant completed the 378


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reaction within 2 h (i.e. 99.1 % conversion and > 99.9 % ee of L-norleucine), while use of the 379

wild-type OATA led to only 24.6 % conversion at 2 h. 380

To demonstrate the catalytic utility of the L57A variant to access production of D-amino 381

acids, we performed kinetic resolution of racemic norvaline to prepare D-norvaline that can be 382

used as a chiral building block of macrolide drugs such as Pamamycin-607 (43) and 383

Epilachnene (44) (Fig. 6C). Starting with 50 mM rac-norvaline and 50 mM glyoxylic acid 384

using the same enzyme concentration as the one in Fig. 6A, the kinetic resolution was 385

completed by the L57A variant within 5 h (i.e. 51.6 % conversion and 99.2 % ee of D-386

norvaline). In contrast, the same reaction catalyzed by the wild-type enzyme led to only 0.8 % 387

conversion and 2.5 % ee at 5 h. 388

CONCLUSIONS 389

To expand the synthetic utility of ω-TAs for production of diverse unnatural amino acids via 390

either asymmetric synthesis or kinetic resolution, it is indispensable to overcome the canonical 391

steric constraint in the small pocket. To the best of our knowledge, this study is the first 392

example to engineer the canonical substrate specificity of a ω-TA for α-keto acids and α-amino 393

acids. Our results indicate that a single point mutation introduced in the large pocket rather than 394

the small pocket could be more effective in relieving the steric constraint. However, a future 395

direction to further engineer the substrate specificity toward more structurally demanding α-396

keto acids should be accompanied by additional mutations in the small pocket of the L57A 397

variant, e.g. the V154A substitution identified beneficial for relieving the steric constraint. 398

399

400


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Supplemental Materials 401

Supplemental Figures S1 - S3. 402

403

Acknowledgements 404

This work was supported by the Advanced Biomass R&D Center (ABC-2011-0031358) 405

through the National Research Foundation of Korea and the R&D grant (S2173394) funded by 406

the Small and Medium Business Administration of Korea. 407


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References 408

1. Patel R. 2013. Biocatalytic synthesis of chiral alcohols and amino acids for 409

development of pharmaceuticals. Biomolecules 3:741-777. 410

2. Gotor-Fernández V, Gotor V. 2009. Biocatalytic routes to chiral amines and amino 411

acids. Curr Opin Drug Discovery Dev 12:784-797. 412

3. Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Sturmer R, Zelinski T. 413

2004. Industrial methods for the production of optically active intermediates. Angew 414

Chem Int Ed 43:788-824. 415

4. Leuchtenberger W, Huthmacher K, Drauz K. 2005. Biotechnological production of 416

amino acids and derivatives: Current status and prospects. Appl Microbiol Biotechnol 417

69:1-8. 418

5. Maier THP. 2003. Semisynthetic production of unnatural L-α-amino acids by 419

metabolic engineering of the cysteine-biosynthetic pathway. Nat Biotechnol 21:422-427. 420

6. Bommarius AS, Schwarm M, Drauz K. 2001. Comparison of different 421

chemoenzymatic process routes to enantiomerically pure amino acids. Chimia 55:50-59. 422

7. Chenault HK, Dahmer J, Whitesides GM. 1989. Kinetic resolution of unnatural and 423

rarely occurring amino acids: Enantioselective hydrolysis of N-acyl amino acids 424

catalyzed by acylase I. J Am Chem Soc 111:6354-6364. 425

8. Krieg L, Ansorge-Schumacher MB, Kula MR. 2002. Screening for amidases: 426

Isolation and characterization of a novel D-amidase from Variovorax paradoxus. Adv 427

Synth Catal 344:965-973. 428


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

21

9. Komeda H, Ishikawa N, Asano Y. 2003. Enhancement of the thermostability and 429

catalytic activity of D-stereospecific amino-acid amidase from Ochrobactrum anthropi 430

SV3 by directed evolution. J Mol Catal B: Enzym 21:283-290. 431

10. Clemente-Jiménez JM, Martínez-Rodríguez S, Rodríguez-Vico F, Heras-Vázquez 432

FJL. 2008. Optically pure α-amino acids production by the "Hydantoinase Process". 433

Recent Pat Biotechnol 2:35-46. 434

11. Altenbuchner J, Siemann-Herzberg M, Syldatk C. 2001. Hydantoinases and related 435

enzymes as biocatalysts for the synthesis of unnatural chiral amino acids. Curr Opin 436

Biotechnol 12:559-563. 437

12. Caligiuri A, D'Arrigo P, Rosini E, Tessaro D, Molla G, Servi S, Pollegioni L. 2006. 438

Enzymatic conversion of unnatural amino acids by yeast D-amino acid oxidase. Adv 439

Synth Catal 348:2183-2190. 440

13. Singh S, Gogoi BK, Bezbaruah RL. 2011. Racemic resolution of some DL-amino 441

acids using Aspergillus fumigatus L-amino acid oxidase. Curr Microbiol 63:94-99. 442

14. Gonçalves LPB, Antunes OAC, Oestreicher EG. 2006. Thermodynamics and kinetic 443

aspects involved in the enzymatic resolution of (R,S)-3-fluoroalanine in a coupled 444

system of redox reactions catalyzed by dehydrogenases. Org Process Res Dev 10:673-445

677. 446

15. Hummel W, Kuzu M, Geueke B. 2003. An efficient and selective enzymatic oxidation 447

system for the synthesis of enantiomerically pure D-tert-Leucine. Org Lett 5:3649-3650. 448

16. Li T, Kootstra AB, Fotheringham IG. 2002. Nonproteinogenic α-amino acid 449

preparation using equilibrium shifted transamination. Org Process Res Dev 6:533-538. 450


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

22

17. Taylor PP, Pantaleone DP, Senkpeil RF, Fotheringham IG. 1998. Novel 451

biosynthetic approaches to the production of unnatural amino acids using transaminases. 452

Trends Biotechnol 16:412-418. 453

18. Stewart JD. 2001. Dehydrogenases and transaminases in asymmetric synthesis. Curr 454

Opin Chem Biol 5:120-129. 455

19. Park ES, Dong JY, Shin JS. 2014. Active site model of (R)-selective ω-transaminase 456

and its application to the production of D-amino acids. Appl Microbiol Biotechnol 457

98:651-660. 458

20. Park ES, Dong JY, Shin JS. 2013. Biocatalytic asymmetric synthesis of unnatural 459

amino acids through the cascade transfer of amino groups from primary amines onto 460

keto acids. ChemCatChem 5:3538-3542. 461

21. Park ES, Dong JY, Shin JS. 2013. ω-Transaminase-catalyzed asymmetric synthesis of 462

unnatural amino acids using isopropylamine as an amino donor. Org Biomol Chem 463

11:6929-6933. 464

22. Park E-S, Malik MS, Dong J-Y, Shin J-S. 2013. One-pot production of enantiopure 465

alkylamines and arylalkylamines of opposite chirality catalyzed by ω-transaminase. 466

ChemCatChem 5:1734-1738. 467

23. Savile CK, Janey JM, Mundorff EC, Moore JC, Tam S, Jarvis WR, Colbeck JC, 468

Krebber A, Fleitz FJ, Brands J, Devine PN, Huisman GW, Hughes GJ. 2010. 469

Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin 470

manufacture. Science 329:305-309. 471


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

23

24. Mutti FG, Fuchs CS, Pressnitz D, Sattler JH, Kroutil W. 2011. Stereoselectivity of 472

four (R)-selective transaminases for the asymmetric amination of ketones. Adv Synth 473

Catal 353:3227-3233. 474

25. Cassimjee KE, Branneby C, Abedi V, Wells A, Berglund P. 2010. Transaminations 475

with isopropylamine: Equilibrium displacement with yeast alcohol dehydrogenase 476

coupled to in situ cofactor regeneration. Chem Comm 46:5569-5571. 477

26. Iwasaki A, Matsumoto K, Hasegawa J, Yasohara Y. 2012. A novel transaminase, 478

(R)-amine:pyruvate aminotransferase, from Arthrobacter sp. KNK168 (FERM BP-479

5228): Purification, characterization, and gene cloning. Appl Microbiol Biotechnol 480

93:1563-1573. 481

27. Shin JS, Kim BG. 2002. Exploring the active site of amine:pyruvate aminotransferase 482

on the basis of the substrate structure-reactivity relationship: How the enzyme controls 483

substrate specificity and stereoselectivity. J Org Chem 67:2848-2853. 484

28. Malik MS, Park ES, Shin JS. 2012. Features and technical applications of ω-485

transaminases. Appl Microbiol Biotechnol 94:1163-1171. 486

29. Park ES, Park SR, Han SW, Dong JY, Shin JS. 2014. Structural determinants for the 487

non-canonical substrate specificity of the ω-transaminase from Paracoccus denitrificans. 488

Adv Synth Catal 356:212-220. 489

30. Midelfort KS, Kumar R, Han S, Karmilowicz MJ, McConnell K, Gehlhaar DK, 490

Mistry A, Chang JS, Anderson M, Villalobos A, Minshull J, Govindarajan S, 491

Wong JW. 2013. Redesigning and characterizing the substrate specificity and activity 492

of Vibrio fluvialis aminotransferase for the synthesis of imagabalin. Protein Eng Des Sel 493

26:25-33. 494


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

24

31. Park ES, Kim M, Shin JS. 2012. Molecular determinants for substrate selectivity of ω-495

transaminases. Appl Microbiol Biotechnol 93:2425-2435. 496

32. Park ES, Shin JS. 2014. Deracemization of amino acids by coupling transaminases of 497

opposite stereoselectivity. Adv Synth Catal 356:3505-3509. 498

33. B'Hymer C, Montes-Bayon M, Caruso JA. 2003. Marfey's reagent: Past, present, and 499

future uses of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. J Sep Sci 26:7-19. 500

34. Bhushan R, Brückner H. 2004. Marfey’s reagent for chiral amino acid analysis: A 501

review. Amino Acids 27:231-247. 502

35. Rausch C, Lerchner A, Schiefner A, Skerra A. 2013. Crystal structure of the ω-503

aminotransferase from Paracoccus denitrificans and its phylogenetic relationship with 504

other class III aminotransferases that have biotechnological potential. Proteins 81:774-505

787. 506

36. Humble MS, Cassimjee KE, Hãkansson M, Kimbung YR, Walse B, Abedi V, 507

Federsel HJ, Berglund P, Logan DT. 2012. Crystal structures of the 508

Chromobacterium violaceum ω-transaminase reveal major structural rearrangements 509

upon binding of coenzyme PLP. FEBS J 279:779-792. 510

37. Fabian Steffen-Munsberg CV, Ahmad Thontowi, Sebastian Sch tzle,, Tony 511

Tumlirsch MSH, Henrik Land, Per Berglund,, Uwe T. Bornscheuer aMH. 2013. 512

Connecting unexplored protein crystal structures to enzymatic function. ChemCatChem 513

5:150-153. 514

38. Han SW, Park ES, Dong JY, Shin JS. 2015. Mechanism-guided engineering of ω-515

transaminase to accelerate reductive amination of ketones. Adv Synth Catal 357:1732-516

1740. 517


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

25

39. Malashkevich VN, Onuffer JJ, Kirsch JF, Jansonius JN. 1995. Alternating arginine-518

modulated substrate specificity in an engineered tyrosine aminotransferase. Nat Struct 519

Mol Biol 2:548-553. 520

40. Cellini B, Bertoldi M, Montioli R, Paiardini A, Borri Voltattorni C. 2007. Human 521

wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: 522

Functional properties and physiological implications. Biochem J 408:39-50. 523

41. Michel V, Jean B, Bernard M, Georges R. 1990. US Patent No. 4,902,817 524

42. Ming XF, Rajapakse AG, Carvas JM, Ruffieux J, Yang Z. 2009. Inhibition of S6K1 525

accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline. 526

BMC Cardiovasc Disord 9: 12. 527

43. Fraser BH, Mulder RJ, Perlmutter P. 2006. The total synthesis of pamamycin-607. 528

Part 2: Synthesis of the C6-C18 domain. Tetrahedron 62:2857-2867. 529

44. Farmer JJ, Attygalle AB, Smedley SR, Eisner T, Meinwald J. 1997. Absolute 530

configuration of insect-produced epilachnene. Tetrahedron Lett 38:2787-2790. 531

532

533

534

535

536


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Table 1. PCR primers used for preparing variants of OATA. 537

Mutation Mutagenesis primersa

Y20A Forward: 5'-CGCGATATCCGTTATCATCTCCATTCTGCGACCGATGCTGTCCG-3' Reverse: 5'-CGGACAGCATCGGTCGCAGAATGGAGATGATAACGGATATCGCG-3'

L57A Forward: 5'-CGAAGCGATGTCAGGAGCGTGGAGTGTTGGCGTG-3' Reverse: 5'-CACGCCAACACTCCACGCTCCTGACATCGCTTCG-3'

L57V Forward: 5'-CGAAGCGATGTCAGGAGTGTGGAGTGTTGGC-3' Reverse: 5'-GCCAACACTCCACACTCCTGACATCGCTTCG-3'

L57G Forward: 5'-TATCGAAGCGATGTCAGGAGGCTGGAGTGTTGGCGTGG-3' Reverse: 5'-CCACGCCAA CACTCCAGCCTCCTGACATCGCTTCGATA-3'

W58A Forward: 5'-GCGATGTCAGGACTGGCGAGTGTTGGCGTGGG-3' Reverse: 5'-CCCACGCCAACACTCGCCAGTCCTGACATCGC-3'

F86A Forward: 5'-AGATGAAGAAGCTGCCTTTCTACCATACAGCGTCCTACCGTTCGCAT-3' Reverse: 5'-ATGCGAACGGTAGGACGCTGTATGGTAGAAAGGCAGCTTCTTCATCT-3'

Y151A Forward: 5'-CTCACGCAAGCGCGGCGCGCACGGTGTGACGATTG-3' Reverse: 5'-CAATCGTCACACCGTGCGCGCCGCGCTTGCGTGAG-3'

V154A Forward: 5'-CGGCTATCACGGTGCGACGATTGCCTCTG-3' Reverse: 5'-CAGAGGCAATCGTCGCACCGTGATAGCCG-3'

I261A Forward: 5'-TCTGCTGATCGCCGACGAGGTTGCGTGCGGCTTCGGA-3' Reverse: 5'-TCCGAAGCCGCACGCAACCTCGTCGGCGATCAGCAGA-3'

F323A Forward: 5'-ACGCTTGGCACGGGCGCGACGGCATCTGGCCAT-3' Reverse: 5'-ATGGCCAGATGCCGTCGCGCCCGTGCCAAGCGT-3'

T324A Forward: 5'-GGCACGGGCTTCGCGGCATCTGGCC-3' Reverse: 5'-GGCCAGATGCCGCGAAGCCCGTGCC-3'

a The mutation sites are underlined.538


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Table 2. Effect of the mutations of the L57 residue on substrate specificity of OATA for α-keto 539

acids. 540

Substrate Side chain Reaction rate (μM/min)a

wild-type L57V L57A L57G

glyoxylic acid -H 618 ± 31 60 ± 2 354 ± 23 113 ± 12

pyruvic acid -CH3 478 ± 25 66 ± 12 367 ± 37 132 ± 6

2-oxobutyric acid -CH2CH3 69 ± 5 13 ± 3 161 ± 6 30 ± 2

2-oxopentanoic acid -(CH2)2CH3 4 ± 2 6 ± 1 190 ± 8 47 ± 4

2-oxohexanoic acid -(CH2)3CH3 2 ± 1 2 ± 1 77 ± 1 10 ± 1

2-oxooctanoic acid -(CH2)5CH3 n.d.b n.d.b 8 ± 4 n.d.b

541

a Reaction rate represents the initial rate per 1 μM enzyme and was measured at 20 mM (S)-α-542

MBA and 20 mM α-keto acid in 50 mM phosphate buffer (pH 7). b not detectable (i.e. reaction 543

rate < 1 μM/min). 544

545

546

547

548


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Table 3. Effect of the mutations of the L57 residue on substrate specificity of OATA for α-549

amino acids. 550

Substrate Side chain Reaction rate (μM/min)a

wild-type L57V L57A L57G

Glycine -H 18 ± 1 3 ± 2 9 ± 4 3 ± 2

L-alanine -CH3 559 ± 4 75 ± 5 203 ± 26 39 ± 5

L-homoalanine -CH2CH3 42 ± 4 2 ± 1 52 ± 1 9 ± 1

L-norvaline -(CH2)2CH3 2 ± 1 n.d.b 111 ± 7 14 ± 1

L-norleucine -(CH2)3CH3 n.d.b n.d.b 26 ± 2 3 ± 1

551

a Reaction rate represents the initial rate per 1 μM enzyme and was measured at 20 mM amino 552

acid and 20 mM propanal in 50 mM phosphate buffer (pH 7). b not detectable (i.e. reaction rate 553

< 1 μM/min). 554

555

556


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Table 4. Kinetic parameters of the wild-type and the L57A OATA for α-keto acids.a 557

α-keto acid wild-type

L57A

KM

(mM)

kcat

(s-1) kcat/KM

(M-1 s-1)

KM

(mM) kcat

(s-1) kcat/KM

(M-1 s-1)

pyruvic acid 0.12 ± 0.01b 2.6 ± 0.1b 22000 ± 3000b

0.34 ± 0.05 3.7 ± 0.2 11000 ± 2000

2-oxohexanoic acid 35 ± 1 0.023 ± 0.001 0.66 ± 0.05 5.7 ± 0.3 1.1 ± 0.1 190 ± 20

a Kinetic parameters represent the apparent rate constants determined at a fixed concentration of (S)-α-MBA. b These kinetic parameters 558

were taken from a previous study (38). 559

560

561

562

563

564

565


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Figure legends. 566

Fig.1 Docking model of L-alanine in the active site of OATA. L-alanine is shown in CPK 567

representation. The internal aldimine, formed between PLP and K287, is shown as 568

thick sticks. K287 is positioned right behind the bound substrate. The cyan dotted lines 569

represent hydrogen bonds. Color use of the active site residues is consistent with that 570

of labels. The active site is visualized by a Connolly surface. 571

Fig.2 Enzyme activities of the alanine scanning mutants for 2-oxopentanoic acid. Reaction 572

rate represents the initial rate per 1 μM enzyme and was measured at 20 mM (S)-α-573

MBA and 20 mM 2-oxopentanoic acid. WT represents the wild-type OATA. 574

Fig. 3 Effect of the mutations of the L57 residue on amino donor activity for isopropylamine. 575

Reaction rate represents the initial rate per 1 μM enzyme and was measured at 20 mM 576

isopropylamine and 20 mM pyruvic acid. 577

Fig. 4 Docking models of the L57A variant using (A) L-alanine and (B) L-norleucine as 578

ligands. The ligands docked in the active site, visualized by a Connolly surface, are 579

represented by ball-and-stick models. The purple sticks and the cyan dotted lines 580

represent the mutation site and the hydrogen bonds, respectively. The yellow sticks in 581

the binding pocket of the upper figure represent the docking pose of L-alanine in the 582

wild-type OATA as shown in Fig. 1. 583

Fig. 5 Effect of the L57A mutation on the enzyme stability. Purified enzymes were incubated 584

in 50 mM potassium phosphate buffer (pH 7.0) at 37 oC. 585


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Fig. 6 Enzymatic reactions to produce unnatural amino acids using the L57A variant in 586

comparison with the parental ω-TA. (A) Asymmetric synthesis of L-norvaline. 587

Reaction conditions were 50 mM 2-oxopentanoic acid, 100 mM isopropylamine and 588

40 μM ω-TA. (B) Asymmetric synthesis of L-norleucine. Reaction conditions were 589

100 mM 2-oxohexanoic acid, 200 mM isopropylamine and 200 μM ω-TA. (C) Kinetic 590

resolution of rac-norvaline. Reaction conditions were 50 mM rac-norvaline, 50 mM 591

glyoxylic acid and 40 μM ω-TA. 592

593

594

595

596

597

598

599

600

601

602

603


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604

605

606

607

608

609

610

611

Fig. 1 612


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613

614

615

WT Y20A L57A W58A F86A Y151A V154A I261A F323A T324A

reac

tion

rate

(μM

/min

)

0

50

100

150

200

616

617

618

619

620

Fig. 2 621


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622

623

624

WT L57V L57A L57G

reac

tion

rate

(μM

/min

)

0

50

100

150

200

250

625

626

627

628

629

Fig. 3 630


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631

632

633

634

Fig. 4 635


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636

637

638

639

Incubation time (h)0 20 40 60 80 100 120

Res

idua

l act

ivity

0.0

0.2

0.4

0.6

0.8

1.0

wild-typeL57A

640

641

642

643

644

645

646

Fig. 5 647


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648

Fig. 6 649


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