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

Abstract

-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...

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

“…Mutagenesis was carried out using a QuikChange Lightning site‐directed mutagenesis kit (Agilent Technologies Co.) according to an instruction manual. All the nine alanine scanning mutants and the double mutant carrying L57A/W58A were constructed in our previous studies . The three mutants carrying L57A/V154A, W58A/V154A and L57A/W58A/V154A mutations were generated by V154A substitution of L57A, W58A and L57A/W58A mutants, respectively, as templates.…”

“…For example, a single point mutation of tryptophan 58 in a ( S )‐selective ω‐TA from Ochrobactrum anthropi (OATA) was proven to dramatically improve catalytic turnover of ketones . Moreover, OATA could be engineered to accept even a n ‐hexyl group of an α‐keto acid in the S pocket by a point mutation of leucine 57 . Nevertheless, we have not succeeded in creating an OATA variant capable of accommodating a branched‐chain alkyl group in the S pocket although protein engineering to accept arylalkyl ketones carrying an isopropyl or a t ‐butyl group was reported elsewhere .…”

Recent Advances in ω-Transaminase-Mediated Biocatalysis for the Enantioselective Synthesis of Chiral Amines

“…This variant allowed the acceptance of bulkier substrates, 2-oxopentanoic acid, and L-norvaline and improved the activity towards these substrates by 48-and 56-fold, respectively. The applicability of the generated mutant was evaluated for the kinetic resolution of racemic norvaline and furthermore to produce optically pure L-and D-norvaline by asymmetric amination of 2-oxopentanoic acid [74]. In another study, Nobili et al carried out systematic mutagenesis study of the active site residues of (S)-ω-TAVf to expand its substrate scope towards two bulky ketones.…”

The ω‐transaminase engineering database (oTAED): A navigation tool in protein sequence and structure space

“…The 3 classification schemes are used in parallel. TAs are named by their stereopreference (( R) /( S )‐selective), their substrate specificity, their regioselectivity (α/ω‐TAs), or their Pfam group (aminotransferase class), resulting in enzyme names such as: ω‐aminotransferase, ( S )‐selective aminotransferase, aromatic amino acid TA, γ‐aminobutyrate TA, ω‐amino acid:pyruvate TAs, and class III aminotransferase . A public online database on ω‐TAs will be a helpful tool, connecting information about mutation sites, structure data and substrate scope, thus allowing researchers the mining of uncharacterized ω‐TAs for the desired enzyme functions and predicting interesting mutation sites.…”