Mechanism‐Guided Engineering of ω‐Transaminase to Accelerate Reductive Amination of Ketones

Han, Shunsheng; Park, Eul Soo; Dong, Joo Young; Shin, Jaeyoung

doi:10.1002/adsc.201500211

Cited by 53 publications

(85 citation statements)

References 49 publications

(30 reference statements)

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“…It is notable that the fold change in k cat is even larger than that in K m , indicating that the L57A-induced alleviation of steric interference in the small pocket benefits the catalytic step much more than the binding step. This result is in accordance with the findings obtained with structural model of a quinonoid intermediate (i.e., the most unstable reaction intermediate), where formation of a covalent linkage between the substrate and PLP rearranges the substrate moiety so that it is close to the PLP side and thereby results in the steric interference of a bulky substituent in the reaction intermediate stronger than that in the Michaelis complex (38).…”

Section: Figsupporting

confidence: 81%

“…Kinetic analysis. A pseudo-one-substrate kinetic model was used to obtain apparent kinetic parameters for pyruvic acid and 2-oxohexanoic acid as described previously (38). The range of pyruvic acid concentrations used for initial rate measurements was 0.5 to 5 mM.…”

Section: Chemicalsmentioning

confidence: 99%

“…a Kinetic parameters represent the apparent rate constants determined at a fixed concentration of (S)-␣-MBA. b These kinetic parameters were taken from a previous study (38).…”

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confidence: 99%

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Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Han

Park

Dong

et al. 2015

Appl Environ Microbiol

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-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 acids. Here we report on the engineering of an (S)-selective -TA from Ochrobactrum anthropi (OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which L-alanine 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-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, 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-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively. U nnatural amino acids are widely used as essential chiral building 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 development of various biocatalytic approaches to afford scalable processes 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 dehydrogenase 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, industrial implementation of the transaminase-mediated synthesis of unnatural amino acids has lagged behind because of low equilibrium constants, usually close to unity, for the reactions between keto acids a...

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Section: Figsupporting

confidence: 81%

Section: Chemicalsmentioning

confidence: 99%

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Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Han

Park

Dong

et al. 2015

Appl Environ Microbiol

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“…Since V NADH levelled off above 5 U mL −1 of ALDH, we set 10 U mL −1 as a working concentration for the ALDH excess condition in further enzyme assays unless V 4 exceeded 32.5 μM min −1 . Note that the ω‐TA used in the enzyme assay was an engineered S ‐selective enzyme from Ochrobactrum anthropi carrying a W58 L substitution (OATA W58L ) which was rationally introduced to impart activity improvements for ketones in the previous study …”

Section: Resultsmentioning

confidence: 99%

“…In the conventional HPLC analysis to measure ω‐TA activity for ketones (e. g., amination of 1 n using alanine or isopropylamine as an amino donor), UV detection of the produced amine (e. g., α‐MBA ( 2 n )) usually permits reliable quantitation of the product formation higher than 10 μM . In this case, an experimental LOD in terms of a reaction rate is over 10 μM min −1 when the reaction is allowed for a minute which is usually regarded as a minimal time required to keep enzyme reactions running reliably.…”

Section: Resultsmentioning

confidence: 99%

Rapid and Quantitative Profiling of Substrate Specificity of ω‐Transaminases for Ketones

Han

Shin

2019

ChemCatChem

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ω‐Transaminases (ω‐TAs) have gained growing attention owing to their capability for asymmetric synthesis of chiral amines from ketones. Reliable high‐throughput activity assay of ω‐TAs is essential in carrying out extensive substrate profiling and establishing a robust screening platform. Here we report spectrophotometric and colorimetric methods enabling rapid quantitation of ω‐TA activities toward ketones in a 96‐well microplate format. The assay methods employ benzylamine, a reactive amino donor for ω‐TAs, as a cosubstrate and exploit aldehyde dehydrogenase (ALDH) as a reporter enzyme, leading to formation of benzaldehyde detectable by ALDH owing to concomitant NADH generation. Spectrophotometric substrate profiling of two wild‐type ω‐TAs of opposite stereoselectivity was carried out at 340 nm with 22 ketones, revealing subtle differences in substrate specificities that were consistent with docking simulation results obtained with cognate amines. Colorimetric readout for naked eye detection of the ω‐TA activity was also demonstrated by supplementing the assay mixture with color‐developing reagents whose color reaction could be quantified at 580 nm. The colorimetric assay was applied to substrate profiling of an engineered ω‐TA for 24 ketones, leading to rapid identification of reactive ketones. The ALDH‐based assay is expected to be promising for high‐throughput screening of enzyme collections and mutant libraries to fish out the best ω‐TA candidate as well as to tailor enzyme properties for efficient amination of a target ketone.

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Combinatorial Mutation Analysis of ω‐Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone

2019

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ω-Transaminase (ω-TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω-TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i. e., Ranalysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active-site residues of ω-TA from Ochrobactrum anthropi (i. e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine-scanning mutants for seven substrate pairs. The R-analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α-keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the Ranalysis-based prediction, the ω-TA variant harboring triple alanine mutations, i. e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e. g. a 3.2 × 10 4 -fold activity increase for 1phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched-chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with > 99% ee of a resulting (S)-amine.

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Mechanism‐Guided Engineering of ω‐Transaminase to Accelerate Reductive Amination of Ketones

Cited by 53 publications

References 49 publications

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Rapid and Quantitative Profiling of Substrate Specificity of ω‐Transaminases for Ketones

Combinatorial Mutation Analysis of ω‐Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone

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