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

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Active-Site Engineering of -Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent Sang-Woo Han, Eul-Soo Park, Joo-Young Dong, Jong-Shik Shin Department of Biotechnology, Yonsei University, Seoul, South Korea -Transaminase (-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most -TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino ac- ids. Here we report on the engineering of an (S)-selective -TA from Ochrobactrum anthropi (OATA) to reduce the steric con- straint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which L-ala- nine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for -keto acids and -amino acids carrying substituents whose bulk is up to that of an n-butyl substituent (e.g., 48- and 56-fold increases in activity for 2-oxopentanoic acid and L-norva- line, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In con- trast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an -carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically pure L- and D-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopan- tanoic acid and kinetic resolution of racemic norvaline, respectively. U nnatural amino acids are widely used as essential chiral build- ing blocks for diverse pharmaceutical drugs, agrochemicals, and chiral ligands (1–3). In contrast to natural amino acids, a fermentative method for the production of unnatural amino acids is not yet commercially available (4, 5). This has driven the devel- opment of various biocatalytic approaches to afford scalable pro- cesses for preparing enantiopure unnatural amino acids. These processes include kinetic resolution of racemic amino acids using acylase (6, 7), amidase (8, 9), hydantoinase (10, 11), and amino acid oxidase (12, 13) and asymmetric reductive amination of keto acids using dehydrogenase (14, 15) and transaminase (16, 17). Asymmetric amination is usually favored over kinetic resolution because the former affords a 100% yield without racemization of an unwanted enantiomer. In contrast to a mandatory requirement for the supply and regeneration of an expensive external cofactor for the dehydroge- nase reactions, transaminase catalyzes the transfer of an amino group from an amino donor to an acceptor using pyridoxal 5=- phosphate (PLP) as a prosthetic group (18). Nevertheless, indus- trial implementation of the transaminase-mediated synthesis of unnatural amino acids has lagged behind because of low equilib- rium constants, usually close to unity, for the reactions between keto acids and amino acids. However, recent studies have demon- strated that the reductive amination of keto acids can be driven to completion without the thermodynamic limitation by employing cheap amines as a cosubstrate when the transfer of an amino group can be mediated by -transaminase (-TA), which utilizes primary amines as an amino donor (19–21). For example, the equilibrium constant for the amination of pyruvic acid by isopro- pylamine was reported to be 67 (22). Therefore, use of a 1.5 molar equivalent of isopropylamine relative to the concentration of py- ruvic acid affords a 97% theoretical conversion of pyruvic acid to L- or D-alanine, depending on the stereoselectivity of -TA. More- over, acetone (i.e., the deamination product of isopropylamine) is highly volatile, and the resulting equilibrium shift by facile evap- oration can drive even thermodynamically unfavorable reactions to completion, as demonstrated elsewhere with the reductive ami- nation of ketones (23–25). Another advantage of using isopropy- lamine as an amino donor is that most -TAs show low levels of activity for acetone (26), which minimizes enzyme inhibition by the ketone product. Taken together, -TA-catalyzed asymmetric amination of keto acids using isopropylamine is a promising strat- egy for the scalable production of unnatural amino acids. One crucial bottleneck for synthesizing diverse unnatural amino acids using -TAs is the very narrow substrate specificity of the enzyme toward keto acids. It is generally accepted that both (R)- and (S)-selective -TAs possess two substrate-binding pock- ets consisting of a large pocket that is capable of dual recognition of hydrophobic and carboxyl groups and a small pocket that can Received 8 May 2015 Accepted 23 July 2015 Accepted manuscript posted online 31 July 2015 Citation Han S-W, Park E-S, Dong J-Y, Shin J-S. 2015. Active-site engineering of -transaminase for production of unnatural amino acids carrying a side chain bulkier than an ethyl substituent. Appl Environ Microbiol 81:6994 –7002. doi:10.1128/AEM.01533-15. Editor: S.-J. Liu Address correspondence to Jong-Shik Shin, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.01533-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.01533-15 6994 aem.asm.org October 2015 Volume 81 Number 20 Applied and Environmental Microbiology on July 26, 2018 by guest http://aem.asm.org/ Downloaded from

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Active-Site Engineering of �-Transaminase for Production ofUnnatural Amino Acids Carrying a Side Chain Bulkier than an EthylSubstituent

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

Department of Biotechnology, Yonsei University, Seoul, South Korea

�-Transaminase (�-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheapamino donors such as isopropylamine. The small substrate-binding pocket of most �-TAs permits entry of substituents nolarger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino ac-ids. Here we report on the engineering of an (S)-selective �-TA from Ochrobactrum anthropi (OATA) to reduce the steric con-straint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which L-ala-nine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants,an L57A variant showed dramatic activity improvements in activity for �-keto acids and �-amino acids carrying substituentswhose bulk is up to that of an n-butyl substituent (e.g., 48- and 56-fold increases in activity for 2-oxopentanoic acid and L-norva-line, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In con-trast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggestedthat the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an �-carboxyl group andthereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out theproduction of optically pure L- and D-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopan-tanoic acid and kinetic resolution of racemic norvaline, respectively.

Unnatural amino acids are widely used as essential chiral build-ing blocks for diverse pharmaceutical drugs, agrochemicals,

and chiral ligands (1–3). In contrast to natural amino acids, afermentative method for the production of unnatural amino acidsis not yet commercially available (4, 5). This has driven the devel-opment of various biocatalytic approaches to afford scalable pro-cesses for preparing enantiopure unnatural amino acids. Theseprocesses include kinetic resolution of racemic amino acids usingacylase (6, 7), amidase (8, 9), hydantoinase (10, 11), and aminoacid oxidase (12, 13) and asymmetric reductive amination of ketoacids using dehydrogenase (14, 15) and transaminase (16, 17).Asymmetric amination is usually favored over kinetic resolutionbecause the former affords a 100% yield without racemization ofan unwanted enantiomer.

In contrast to a mandatory requirement for the supply andregeneration of an expensive external cofactor for the dehydroge-nase reactions, transaminase catalyzes the transfer of an aminogroup from an amino donor to an acceptor using pyridoxal 5=-phosphate (PLP) as a prosthetic group (18). Nevertheless, indus-trial implementation of the transaminase-mediated synthesis ofunnatural amino acids has lagged behind because of low equilib-rium constants, usually close to unity, for the reactions betweenketo acids and amino acids. However, recent studies have demon-strated that the reductive amination of keto acids can be driven tocompletion without the thermodynamic limitation by employingcheap amines as a cosubstrate when the transfer of an aminogroup can be mediated by �-transaminase (�-TA), which utilizesprimary amines as an amino donor (19–21). For example, theequilibrium constant for the amination of pyruvic acid by isopro-pylamine was reported to be 67 (22). Therefore, use of a 1.5 molarequivalent of isopropylamine relative to the concentration of py-ruvic acid affords a 97% theoretical conversion of pyruvic acid to

L- or D-alanine, depending on the stereoselectivity of �-TA. More-over, acetone (i.e., the deamination product of isopropylamine) ishighly volatile, and the resulting equilibrium shift by facile evap-oration can drive even thermodynamically unfavorable reactionsto completion, as demonstrated elsewhere with the reductive ami-nation of ketones (23–25). Another advantage of using isopropy-lamine as an amino donor is that most �-TAs show low levels ofactivity for acetone (26), which minimizes enzyme inhibition bythe ketone product. Taken together, �-TA-catalyzed asymmetricamination of keto acids using isopropylamine is a promising strat-egy for the scalable production of unnatural amino acids.

One crucial bottleneck for synthesizing diverse unnaturalamino acids using �-TAs is the very narrow substrate specificity ofthe enzyme toward keto acids. It is generally accepted that both(R)- and (S)-selective �-TAs possess two substrate-binding pock-ets consisting of a large pocket that is capable of dual recognitionof hydrophobic and carboxyl groups and a small pocket that can

Received 8 May 2015 Accepted 23 July 2015

Accepted manuscript posted online 31 July 2015

Citation Han S-W, Park E-S, Dong J-Y, Shin J-S. 2015. Active-site engineering of�-transaminase for production of unnatural amino acids carrying a side chainbulkier than an ethyl substituent. Appl Environ Microbiol 81:6994 –7002.doi:10.1128/AEM.01533-15.

Editor: S.-J. Liu

Address correspondence to Jong-Shik Shin, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01533-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01533-15

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accept substituents no larger than an ethyl group (27, 28). Thecanonical structural features of the active site of �-TAs render acarboxyl group and a side chain of keto acid substrates bound tothe large and small pockets, respectively. This leads all known�-TAs to accept a limited range of �-keto acids, such as glyoxylicacid, pyruvic acid, and 2-oxobutyric acid. The only exception tothe canonical substrate specificity to date is an (S)-selective �-TAfrom Paracoccus denitrificans that can accommodate substituentswhose bulk is up to that of an n-butyl substituent of �-keto acid inthe small pocket (29). We demonstrated that a single point muta-tion in the small pocket (i.e., V153A) endowed the �-TA withsubstantial activity toward even 2-oxooctanoic acid carrying ann-hexyl side chain (29). Despite the noncanonical steric constraintof the �-TA from P. denitrificans, a low level of enzyme activity forisopropylamine renders a synthetic potential of the �-TA less op-timal for the practical amination of the bulky keto acids (21). Thisled us to set out to engineer the substrate specificity of the canon-ical �-TAs showing a high level of activity for isopropylamine. Tothis end, we chose an (S)-selective �-TA from Ochrobactrum an-thropi (OATA), which showed 43% of the activity for isopropyl-amine relative to its activity for (S)-�-methylbenzylamine [(S)-�-MBA] (21).

To date, there have been three reports dealing with engineeringof the canonical steric constraint in the small pocket of �-TAs. Inthe first example, an (R)-selective �-TA from an Arthrobacter sp.was successfully engineered to create a variant that can acceptbulky arylalkyl ketones as an amino acceptor (23). In our previousstudy, the substrate specificity of the engineered variant, harbor-ing 27 amino acid substitutions, was examined using structurallydiverse �-keto acids (21). In spite of the remarkable improve-ments in activity for bulky arylalkyl ketones, the canonical stericconstraint of a parental enzyme toward �-keto acids was not sig-nificantly altered in the engineered variant; e.g., the activity for2-oxopentanoic acid was only 2% of that for pyruvic acid. Thisresult suggests that engineering of the steric constraint should beguided in the context of a substrate type, especially substituentsbound to the large pocket (i.e., hydrophobic versus carboxyl). Thesecond example was carried out with an (S)-selective �-TA fromVibrio fluvialis to improve the activity toward a �-keto ester [i.e.,(R)-ethyl-5-methyl 3-oxooctanoate] (30). The resulting variant,carrying 8 amino acid substitutions, showed a 60-fold increase inactivity for the target �-keto ester, but the substitutions led to acomplete loss of activity for keto acids. The third example em-ployed the same �-TA as the one used in the second example andfocused on improving activities for arylalkyl ketones. To the bestof our knowledge, active-site engineering of a �-TA to relieve thecanonical steric constraint for �-keto acids and �-amino acids hasnot yet been reported.

In this study, we aimed to create a variant of OATA capableof accommodating bulky �-keto acids. To this end, we per-formed substrate docking simulations to select key active-siteresidues and carried out alanine scanning mutagenesis to iden-tify a hot spot responsible for the narrow substrate specificityfor �-keto acids. The resulting variant, carrying only a singlepoint mutation in the large pocket, showed a desirable relax-ation of the canonical steric constraint and afforded efficientstereoselective amination of bulky �-keto acids using isopro-pylamine as an amino donor.

MATERIALS AND METHODSChemicals. Pyruvic acid was obtained from Kanto Chemical Co. (Tokyo,Japan). Isopropylamine was purchased from Junsei Chemical Co. (Tokyo,Japan). L-Alanine was purchased from Acros Organics Co. (Geel, Bel-gium). All other chemicals were purchased from Sigma-Aldrich Co. (St.Louis, MO). Materials used for the preparation of the culture media,including yeast extract, tryptone, and agar, were purchased from BD Bio-sciences (Franklin Lakes, NJ).

Site-directed mutagenesis of �-TA. Single point mutations of OATAwere carried out using a QuikChange Lightning site-directed mutagenesiskit (Agilent Technologies Co., Santa Clara, CA) according to the guide-lines in the instruction manual. The template used for mutagenesis PCRwas pET28-OATA, which was previously constructed (31). Mutagenesisprimers were designed by a primer design program (Agilent). The primersequences are provided in Table 1. The intended mutagenesis was con-firmed by DNA sequencing.

Expression and purification of �-TAs. Overexpression of His6-tagged �-TAs was carried out as described previously with minor modi-fications (32). Escherichia coli BL21(DE3) cells carrying the expressionvector [i.e., pET28a(�) harboring the �-TA gene] were cultivated in LBmedium (typically, 1 liter) containing 50 �g/ml kanamycin. Protein ex-pression was induced by IPTG (isopropyl-�-D-thiogalactopyranoside) atan optical density at 600 nm of 0.4, and the cells were allowed to grow for10 h. The culture broth was centrifuged, and the resulting cell suspensionwas subjected to ultrasonic disruption. Protein purification was carriedout as described previously (32). Protein purity was confirmed by SDS-PAGE (see Fig. S1 in the supplemental material). The molar concentra-tions of the purified �-TAs were determined by measuring the UV absor-bance at 280 nm.

Enzyme assay. All enzyme assays were carried out at 37°C and pH 7(50 mM phosphate buffer). Unless otherwise specified, the reaction con-ditions for the activity assay were 10 mM (S)-�-MBA and 10 mM pyruvicacid. The typical reaction volume was 50 �l. The enzyme reaction wasstopped after 10 min by adding 300 �l acetonitrile. The acetophenoneproduced from the reactions was analyzed by high-pressure liquid chro-matography (HPLC). For initial rate measurements, reaction conversionswere limited to less than 10%.

Substrate specificity. To examine the substrate specificity of theOATA variants, initial rate measurements (i.e., �10% conversion) wereindependently obtained in triplicate. The reaction conditions for exami-nation of the substrate specificity for amino acceptors were 20 mM �-ketoacid and 20 mM (S)-�-MBA in 50 mM phosphate buffer (pH 7). Theacetophenone produced was analyzed by HPLC. To measure activities for�-amino acids, 20 mM amino donor and 20 mM propanal were used asthe substrates, and the �-keto acids produced were analyzed by HPLC. Tomeasure amino donor activities for isopropylamine, reaction conditionswere 20 mM isopropylamine and 20 mM pyruvic acid. L-Alanine wasanalyzed by chiral HPLC after derivatization with Marfey’s reagent(33, 34).

Molecular modeling. Using four X-ray structures of (S)-selective�-TAs as the templates, an homology model of OATA was constructed byuse of the Modeler module (version 9.8) of the Discovery Studio package(version 3.5.0; BIOVIA, San Diego, CA). The X-ray structures used as thetemplates were �-TAs from P. denitrificans (PDB accession number4GRX) (35), Chromobacterium violaceum (PDB accession number 4A6T)(36), Mesorhizobium loti (PDB accession number 3GJU) (37), and Rho-dobacter sphaeroides (PDB accession number 3I5T) (37). The outward-pointing arginine of the structure with PDB accession number 4GRX wasset to be conserved in the homology model, resulting in a dimeric struc-ture of OATA in which each subunit has a different conformation of theactive-site arginine. To construct a holoenzyme structure, the PLP moietywas copied from the structure with PDB accession number 4A6T. Only0.4% nonglycine residues were found by a Ramachandran phi-psi analysisto lie in the disallowed region.

The active-site models of the L57A and L57G variants were built by

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amino acid substitution and then energy minimization (2,000 steps; di-electric constant � 4) of the mutation site, the internal aldimine, and theneighboring residues within 3 Å from L57 (i.e., S55, G56, L57, W58, S59,F82, H84, and T324) until the root mean squared gradient reached 0.1kcal/mol/Å.

Docking simulations with L-alanine and L-norleucine as ligands wereaccomplished using the CDOCKER module under a default setting withinthe active site defined by the Binding-Site module. The most stable dock-ing pose was chosen as a docking model.

Kinetic analysis. A pseudo-one-substrate kinetic model was used toobtain apparent kinetic parameters for pyruvic acid and 2-oxohexanoicacid as described previously (38). The range of pyruvic acid concentra-tions used for initial rate measurements was 0.5 to 5 mM. The ranges of2-oxohexanoic acid concentrations were 7 to 200 mM and 1 to 30 mM forthe wild type and the L57A variant, respectively. The concentration of thecosubstrate [i.e., (S)-�-MBA] was fixed at 20 mM. The acetophenoneproduced was analyzed by HPLC to measure the initial rates. The initialrate data were fitted to a Michaelis-Menten equation, and the Km and kcat

values were calculated from the slopes and y intercepts of the double-reciprocal plots.

Measurement of enzyme stability. To examine the effect of the L57Amutation on enzyme stability, 10 �M enzyme was incubated in 50 mMpotassium phosphate buffer (pH 7) at 37°C. Aliquots of the enzyme solu-tion were sampled at predetermined incubation times and were mixedwith reaction buffer containing 10 mM pyruvic acid and 10 mM (S)-�-MBA. Initial reaction rates were measured by analyzing the acetophenoneproduced. Inactivation constants were obtained by curve fitting of theresidual activity data to a single exponential function.

Enzyme reactions to produce unnatural amino acids. The reactionvolume for the enzyme reactions was 1 ml, and the reaction mixture in 50

mM potassium phosphate buffer (pH 7) was incubated at 37°C. The re-action conditions for the asymmetric synthesis of L-norvaline were 50 mM2-oxopentanoic acid, 100 mM isopropylamine, and 40 �M �-TA. Thesynthesis of L-norleucine was carried out under the reaction conditions of100 mM 2-oxohexanoic acid, 200 mM isopropylamine, 0.1 mM PLP, and

FIG 1 Model of L-alanine docking in the active site of OATA. L-Alanine isshown in Corey-Pauling-Koltun (CPK) representation. The internal aldimine,formed between PLP and K287, is shown as thick sticks. K287 is positionedright behind the bound substrate. The cyan dotted lines represent hydrogenbonds. The colors used for the active-site residues are consistent with thecolors of the labels. The active site is visualized by use of a Connolly surface.

TABLE 1 PCR primers used for preparing variants of OATA

Mutation Orientation 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.

Han et al.

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200 �M �-TA. The kinetic resolution of racemic norvaline (rac-norva-line) was performed with 50 mM rac-norvaline, 50 mM glyoxylic acid, and40 �M �-TA. Aliquots of the reaction mixture (50 �l) were taken atpredetermined reaction times and mixed with 10 �l HCl solution (5 N) tostop the reaction. The reaction mixtures were subjected to HPLC analysisfor measurement of conversion and enantiomeric excess.

HPLC analysis. All HPLC analyses were performed on a Waters HPLCsystem (Milford, MA). Analysis of acetophenone was performed using aSunfire C18 column (Waters Co.) with isocratic elution with 60% metha-nol– 40% water– 0.1% trifluoroacetic acid at 1 ml/min. UV detection wasdone at 254 nm. Quantitative chiral analyses of alanine, norvaline, andnorleucine were carried out using a Crownpak CR() column (DaicelCo., Japan) or a Sunfire C18 column after chiral derivatization with Mar-fey’s reagent (33, 34). eto acids were analyzed using an Aminex HPX-87H column (Bio-Rad, Hercules, CA) with isocratic elution of a 5 mMH2SO4 solution at 0.5 ml/min. The column oven temperature was set to40°C, and UV detection was done at 210 nm.

RESULTS AND DISCUSSIONSubstrate docking. We set out to construct a homology model ofOATA to identify key active-site residues responsible for rejectingthe entry of bulky substituents in the small pocket. The X-raystructures used as the templates for the homology modeling were�-TAs from P. denitrificans (PDB accession number 4GRX; se-quence identity, 41%) (35), C. violaceum (PDB accession number4A6T; sequence identity, 41%) (36), M. loti (PDB accession num-ber 3GJU; sequence identity, 43%) (37), and R. sphaeroides (PDB

accession number 3I5T; sequence identity, 34%) (37). The align-ment of the OATA sequence with the sequences of the template�-TAs is shown in Fig. S2 in the supplemental material. The fourtemplate �-TAs adopt a homodimeric structure where both ac-tive-site arginines assume an inward conformation (i.e., theypoint toward the large pocket), except for the structure with PDBaccession number 4GRX, where one subunit harbors an outwardarginine (i.e., it points toward a solvent side). It is known that theactive-site arginine, responsible for recognition of a carboxylate ofan incoming substrate, undergoes a gross movement and assumesthe outward conformation when the large pocket is taken up by ahydrophobic substituent (39).

We performed substrate docking simulations using the sub-unit structure where the active-site arginine (i.e., R417) adopts theinward conformation (Fig. 1). For the docking simulation, weused L-alanine, instead of pyruvic acid, as a ligand because all theactive-site lysines in the template �-TAs form a Schiff base withPLP (i.e., an internal aldimine capable of deamination of an aminodonor), and thereby, the resulting homology model adopts theinternal aldimine structure. It is notable that the binding of L-al-anine is coordinated by a hydrogen bond between the aminogroup of the substrate and the phenolic oxygen of the PLP moietyas well as multiple hydrogen bonds between the �-carboxylate andthe active-site arginine. Compared to the active-site structure,where R417 adopts an outward conformation, the large pocketbecomes contracted owing to the inward conformation of R417 torecognize the carboxyl group. The small pocket accommodatingthe methyl group of L-alanine is wrapped by side chains of the fiveactive-site residues (i.e., Y20, F86, Y151, V154, and T324), a phos-phate group of the PLP, and a backbone chain ranging from G322to T324. The Connolly surface of the active site clearly explainswhy the small pocket can accept a substituent whose bulk is onlyup to that of an ethyl substituent.

Alanine scanning mutagenesis of the active-site residues.The docking model indentified 13 active-site residues that partic-ipate in the formation of the active-site surface and lie within a 7-Ådistance from the bound L-alanine (i.e., Y20, L57, W58, F86, Y151,V154, A230, I261, K287, G322, F323, T324, and R417). We carriedout alanine substitution of the active-site residues one by one toexamine which residue could be reduced in size to lead to thegeneration of the additional room required for bulky substituentsto be accepted in the small pocket. A230 and G322 were excludedfrom the mutagenesis because alanine substitution of the two res-idues would not generate more room in the active site. K287 andR417 were also excluded because these are essential residues re-sponsible for catalytic turnover and recognition of a carboxyl

FIG 2 Enzyme activities for 2-oxopentanoic acid of the alanine mutants thatwere scanned. The reaction rate represents the initial rate per 1 �M enzymeand was measured with 20 mM (S)-�-MBA and 20 mM 2-oxopentanoic acid.WT, wild-type OATA.

TABLE 2 Effect of mutations of L57 residue on substrate specificity of OATA for �-keto acids

Substrate Side chain

Reaction rate (�M/min)a

Wild type L57V variant L57A variant L57G variant

Glyoxylic acid OH 618 � 31 60 � 2 354 � 23 113 � 12Pyruvic acid OCH3 478 � 25 66 � 12 367 � 37 132 � 62-Oxobutyric acid OCH2CH3 69 � 5 13 � 3 161 � 6 30 � 22-Oxopentanoic acid O(CH2)2CH3 4 � 2 6 � 1 190 � 8 47 � 42-Oxohexanoic acid O(CH2)3CH3 2 � 1 2 � 1 77 � 1 10 � 12-Oxooctanoic acid O(CH2)5CH3 NDb ND 8 � 4 NDa The reaction rate represents the initial rate per 1 �M enzyme and was measured with 20 mM (S)-�-MBA and 20 mM �-keto acid in 50 mM phosphate buffer (pH 7).b ND, not detectable (i.e., the reaction rate was �1 �M/min).

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group, respectively. Therefore, nine active-site residues were sub-jected to alanine scanning mutagenesis, and the enzyme activitiesof the resulting variants toward 2-oxopentanoic acid were mea-sured (Fig. 2). Among the nine variants, an L57A variant showedan exceptionally improved activity for 2-oxopentanoic acid (i.e., a48-fold increase in activity relative to that of the wild-type en-zyme). This was an unexpected result because the L57 residueparticipates in the large pocket. Besides the L57A variant, theV154A variant was the only alanine substitution variant that al-lowed an improvement in activity for 2-oxopentanoic acid (i.e., a3-fold increase). This result is in line with our previous observa-tion that an alanine substitution at the same position of the �-TAfrom P. denitrificans achieved excavation of the small pocket (29).

Site-directed mutagenesis of L57. We presumed that the re-markable improvement in activity for 2-oxopentanoic acidachieved with the L57A mutation would result from altered bind-ing of a carboxyl group owing to the generation of more room inthe large pocket because of the reduction in the size of L57. Toexamine whether a greater improvement in activity could be in-duced by substitution of L57 with small amino acids other thanalanine, we prepared L57V and L57G variants by site-directedmutagenesis. Enzyme activities for various �-keto acids were mea-sured and compared with those of the wild-type enzyme and theL57A variant (Table 2). The wild-type OATA showed negligibleactivities (i.e., �1% relative to that for pyruvic acid) for bulky�-keto acids carrying a side chain larger than an ethyl group. TheL57V mutation did not induce any improvement in activity for thebulky �-keto acids but caused drastic losses of activity towardnative substrates (i.e., substrates whose bulks ranged from thebulk of glyoxylic acid to the bulk of 2-oxobutyric acid). In con-trast, the L57A variant showed improvements in activity for�-keto acids carrying side chains larger than a methyl group (i.e.,2-, 48-, and 39-fold increases in activity for 2-oxobutyric acid,2-oxopentanoic acid, and 2-oxohexanoic acid, respectively). Theimprovements in activity for bulky �-keto acids were so remark-able that the L57A variant exhibited substantial activity even for2-oxooctanoic acid (i.e., 2% relative to that for pyruvic acid). TheL57G substitution also induced improvements in activity forbulky �-keto acids but to much less of a degree than those ob-served with the L57A substitution. Despite the improvements inactivity toward �-keto acids carrying bulky linear alkyl groups,neither the L57A variant nor the L57G variant displayed substan-tial activities for branched-chain �-keto acids, including 3-meth-yl-2-oxobutyric acid, 3-methyl-2-oxopentanoic acid, 4-methyl-2-oxopentanoic acid, and trimethylpyruvic acid (data not shown).

As shown in Table 3, the relaxed steric constraint for �-ketoacids was also observed for �-amino acids (i.e., 56- and 5-fold

improvements in the activities of the L57A and L57G variants,respectively, for L-norvaline). Despite the nondetectable activityof the wild-type OATA for L-norleucine, the relaxed steric con-straint allowed the L57A and L57G variants to show substantialactivities for L-norleucine. The L57V mutation led to drastic de-creases in activity for �-amino acids, as was also observed with�-keto acids. Interestingly, all the OATA variants, in addition tothe wild type, showed very low activities for glycine, despite thehigh levels of activity for the cognate keto acid. These extremelyopposite activities for glycine and glyoxylic acid seem to be ascrib-able to the thermodynamically unfavorable conversion of glycineto its keto acid (40). As was observed with the branched-chain�-keto acids, both the L57A and the L57G variants showednondetectable activities for �-amino acids carrying branched-chain substituents, including L-valine, L-leucine, L-isoleucine, andL-tert-leucine (data not shown).

The synthetic utility of the L57A variant for the asymmetricamination of bulky keto acids is contingent upon a high level ofactivity for isopropylamine as well as no loss of stereoselectivity inthe parent. We measured the amino donor reactivity of isopropy-lamine with the L57 variants (Fig. 3). In contrast to the findings forthe L57V and L57G variants, the L57A variant conserved 80% ofthe parental activity for isopropylamine. In addition to the wild-type OATA, all L57 variants showed the nondetectable formationof D-alanine during the transamination between isopropylamineand pyruvic acid (i.e., enantiomeric excess [ee] of L-alanine pro-

TABLE 3 Effect of mutations of L57 residue on substrate specificity of OATA for �-amino acids

Substrate Side chain

Reaction rate (�M/min)a

Wild type L57V variant L57A variant L57G variant

Glycine OH 18 � 1 3 � 2 9 � 4 3 � 2L-Alanine OCH3 559 � 4 75 � 5 203 � 26 39 � 5L-Homoalanine OCH2CH3 42 � 4 2 � 1 52 � 1 9 � 1L-Norvaline O(CH2)2CH3 2 � 1 NDb 111 � 7 14 � 1L-Norleucine O(CH2)3CH3 ND ND 26 � 2 3 � 1a The reaction rate represents the initial rate per 1 �M enzyme and was measured with 20 mM amino acid and 20 mM propanal in 50 mM phosphate buffer (pH 7).b ND, not detectable (i.e., the reaction rate was �1 �M/min).

FIG 3 Effect of mutations of the L57 residue on amino donor activity forisopropylamine. The reaction rate represents the initial rate per 1 �M enzymeand was measured with 20 mM isopropylamine and 20 mM pyruvic acid.

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duced, �99.9%). This result indicates that the three amino acidsubstitutions of L57 do not affect the stringent stereoselectivity ofthe parental enzyme.

Structural modeling of the L57A variant. To provide struc-tural insight into how the L57A mutation relieves the steric con-straint in the small pocket, we performed a docking simulationwith the L57A variant using L-alanine as a ligand (Fig. 4A). Com-pared to the docking pose of L-alanine in the wild-type active site,the L57A mutation allowed docking of L-alanine slightly awayfrom the small pocket with 0.71- and 0.73-Å translocations of theC-� and C-� of the substrate to the large pocket, respectively.These translocations are attributed to a 1.35-Å movement of the�-carboxyl carbon to the W58 residue owing to the room createdby the L57A substitution. The altered binding orientation of the�-carboxylate in the large pocket permits the formation of anadditional hydrogen bond with the indole group of W58, whichleads to a concurrent loss of one of the preexisting hydrogen bondswith R417.

The altered binding of the �-carboxylate in the active site of theL57A variant led to the substrate docking closer to the large

pocket, which provides more effective room to the small pocketeven without any structural change in the small pocket. In thesubstrate specificity experiments, we could test neither �-ketoacid nor �-amino acid substrates carrying an n-pentyl side chainbecause these substrates were not commercially available. To ex-amine how large of a substituent that the extended small pocketcan accommodate, we carried out a docking simulation using L-nor-leucine as a ligand (Fig. 4B). The findings obtained with the dockingmodel are consistent with the significant enzyme activity for L-nor-leucine and predict that the small pocket cannot accept an n-pentylgroup. The n-butyl group of the bound L-norleucine does not assumea staggered conformation due to a steric interference with the resi-dues of the small pocket, resulting in an intramolecular steric strain ofthe bound substrate. This could explain the 87% loss of activity forL-norleucine compared to that for L-alanine.

Contrary to expectation, the L57G variant did not show higheractivities toward bulky substrates than the L57A variant did. Toexamine how much the L57G mutation allowed the altered bind-ing of the �-carboxyl group to the large pocket, we performed adocking simulation with L-norleucine in the active site of the L57Gvariant (see Fig. S3 in the supplemental material). The dockingpose of L-norleucine in the L57G variant turned out to be veryclose to that generated with the L57A variant, indicating that cre-ation of more room in the large pocket by the L57G mutation didnot elicit a greater translocation of the �-carboxyl group towardthe large pocket. This is in agreement with the findings obtainedwith the model of L-norleucine docking with the L57A variant,where the �-carboxyl group is already in a close contact with W58,and thereby, further translocation of the �-carboxyl group is pro-hibited because of a steric clash against W58 (Fig. 4B). It is pre-sumed that the lower activities of the L57G variant for bulky sub-strates compared to those of the L57A variant result from anincreased conformation flexibility of the backbone chain, whichmight be deleterious to a catalytic step after the formation of aMichaelis complex. This is supported by the drastic losses of ac-tivity of the L57G variant even for the native substrates, such asglyoxylic acid, pyruvic acid, and L-alanine. Note that two consec-utive glycine residues happened to be created immediately beforeW58 in the L57G variant because the 56th residue is glycine (seeFig. S2 in the supplemental material).

Kinetic analysis of the L57A variant. To provide a mechanisticunderstanding of how the L57A mutation improves activities forbulky substrates, we compared the kinetic parameters of the wild-type and the L57A variant for pyruvic acid and 2-oxohexanoicacid (Table 4). Compared with the specificity constant (i.e., kcat/Km) of the wild-type enzyme for pyruvic acid, the L57A variantshowed a 50% reduction in the specificity constant. This loss ofactivity for the native substrate was caused by a 2.8-fold decreasein binding affinity, although the L57A mutation induced a 1.4-fold increase in the turnover number. The weakened binding topyruvic acid by the variant with the L57A mutation seems to resultfrom the loss of a native hydrogen bond between R417 and the�-carboxylate of pyruvic acid (Fig. 1 and 4B).

As the size of the side chain of the �-keto acid substrate extendsfrom that of a methyl group to that of an n-butyl group (i.e., fromthe side chain found in pyruvic acid to that found in 2-oxo-hexanoic acid), the wild-type enzyme showed 290- and 110-folddecreases in binding affinity and the turnover number, respec-tively. The deterioration in both the binding and the catalytic stepsrenders the wild-type enzyme nearly inactive toward 2-oxo-

FIG 4 Docking models of the L57A variant using L-alanine (A) and L-norleu-cine (B) as ligands. The ligands that docked in the active site, visualized by useof a Connolly surface, are represented by ball-and-stick models. Purple sticksand cyan dotted lines, the mutation site and the hydrogen bonds, respectively;yellow sticks in the binding pocket in panel A, the docking pose of L-alanine inthe wild-type OATA, as shown in Fig. 1.

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hexanoic acid. The L57A mutation turned out to promote bothsteps (i.e., 6- and 48-fold increases in binding affinity and catalyticturnover, respectively), leading to a 290-fold increase in the spec-ificity constant for 2-oxohexanoic acid. It is notable that the foldchange in kcat is even larger than that in Km, indicating that theL57A-induced alleviation of steric interference in the small pocketbenefits the catalytic step much more than the binding step. Thisresult is in accordance with the findings obtained with structuralmodel of a quinonoid intermediate (i.e., the most unstable reac-tion intermediate), where formation of a covalent linkage betweenthe substrate and PLP rearranges the substrate moiety so that it isclose to the PLP side and thereby results in the steric interferenceof a bulky substituent in the reaction intermediate stronger thanthat in the Michaelis complex (38).

Synthetic utility of the L57A variant. For the practical appli-cability of enzyme variants engineered to attain a desirable func-tionality, it is essential for the amino acid substitution to disturbthe protein stability in a minimal way. We performed time coursemonitoring of the enzyme activities under the incubation condi-tions of 50 mM phosphate buffer (pH 7.0) and 37°C, which werethe same as those used with the reaction mixture used for prepar-ative purposes (Fig. 5). Intriguingly, we found that the L57A mu-tation remarkably increased the stability of the enzyme. The inac-tivation constants obtained from curve fitting of the residualactivity data to a single exponential function were 1.1 102 h1

(r2 � 0.97, half-life � 62 h) and 8.1 104 h1 (r2 � 0.89, half-life � 860 h) for the wild type and the L57A variant, respectively.The striking improvement in stability renders the L57A varianthighly promising for use in industrial applications.

To examine how the L57A mutation improves the catalyticpotential of OATA for the production of bulky unnatural aminoacids, we carried out the asymmetric synthesis of L-norvaline,which is a key intermediate of perindopril (i.e., an angiotensin-converting enzyme inhibitor) (41) and a potential inhibitor ofarginase (42). As expected, the L57A variant afforded a muchfaster synthesis of L-norvaline from 50 mM 2-oxopentanoic acidand 100 mM isopropylamine than its parental enzyme did underthe same reaction conditions (Fig. 6A). The level of conversionreached 99.3% at 2 h, with the ee of the resulting L-norvalineobtained using the L57A variant being �99.9%, whereas the wild-type OATA permitted only 22.6% conversion at 2 h. As shown inFig. 6B, we also performed the asymmetric synthesis of L-norleu-

FIG 5 Effect of the L57A mutation on enzyme stability. Purified enzymes wereincubated in 50 mM potassium phosphate buffer (pH 7.0) at 37°C.

FIG 6 Enzymatic reactions to produce unnatural amino acids by use of the L57Avariant in comparison with the use of parental �-TA. (A) Asymmetric synthesis ofL-norvaline. Reaction conditions were 50 mM 2-oxopentanoic acid, 100 mM iso-propylamine, and 40 �M �-TA. (B) Asymmetric synthesis of L-norleucine. Reac-tion conditions were 100 mM 2-oxohexanoic acid, 200 mM isopropylamine, and200 �M �-TA. (C) Kinetic resolution of rac-norvaline. Reaction conditions were50 mM rac-norvaline, 50 mM glyoxylic acid, and 40 �M �-TA.

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cine from 100 mM 2-oxohexanoic acid and 200 mM isopropyl-amine using an enzyme concentration 5-fold higher than thatused in the assay whose results are presented in Fig. 6A. The L57Avariant completed the reaction within 2 h (i.e., 99.1% conversionand a �99.9% ee of L-norleucine), while use of the wild-typeOATA led to only 24.6% conversion at 2 h.

To demonstrate the catalytic utility of the L57A variant for theproduction of D-amino acids, we performed the kinetic resolutionof racemic norvaline to prepare D-norvaline for use as a chiralbuilding block of macrolide drugs, such as pamamycin-607 (43)and epilachnene (44) (Fig. 6C). Starting with 50 mM rac-norva-line and 50 mM glyoxylic acid using the same enzyme concentra-tion as the one used in the reaction whose results are presented inFig. 6A, the kinetic resolution was completed by the L57A variantwithin 5 h (i.e., 51.6% conversion and a 99.2% ee of D-norvaline).In contrast, the same reaction catalyzed by the wild-type enzymeled to only 0.8% conversion and a 2.5% ee at 5 h.

Conclusions. To expand the synthetic utility of �-TAs for theproduction of diverse unnatural amino acids via either asymmet-ric synthesis or kinetic resolution, it is indispensable to overcomethe canonical steric constraint in the small pocket. To the best ofour knowledge, this study is the first example of the engineering ofthe canonical substrate specificity of a �-TA for �-keto acids and�-amino acids. Our results indicate that a single point mutationintroduced in the large pocket rather than the small pocket couldbe more effective in relieving the steric constraint. However, fur-ther engineering of the substrate specificity toward more structur-ally demanding �-keto acids should include additional mutationsin the small pocket of the L57A variant, e.g., the V154A substitu-tion identified to be beneficial for relieving the steric constraint.

ACKNOWLEDGMENTS

This work was supported by the Advanced Biomass R&D Center (ABC-2011-0031358) through the National Research Foundation of Korea andan R&D grant (S2173394) funded by the Small and Medium BusinessAdministration of Korea.

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TABLE 4 Kinetic parameters of the wild-type and the L57A OATA for �-keto acidsa

�-Keto acid

Wild type L57A variant

Km (mM) kcat (s1) kcat/Km (M1 s1) Km (mM) kcat (s1) kcat/Km (M1 s1)

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