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

Hong Gon Kim, Sang Woo Han, jong shik shin

Research output: Contribution to journalArticle

Abstract

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., R-analysis) 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 R-analysis-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 3.2×104-fold activity increase for 1-phenylbutylamine. 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. (Figure presented.).

Original languageEnglish
Pages (from-to)2594-2606
Number of pages13
JournalAdvanced Synthesis and Catalysis
Volume361
Issue number11
DOIs
Publication statusPublished - 2019 Jun 6

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Amination
Transaminases
Substrates
Ketones
Alanine
Amines
Enzymes
Keto Acids
Valine
Leucine
Tryptophan
Scanning
Acids
Chemical analysis

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Organic Chemistry

Cite this

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title = "Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone",
abstract = "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., R-analysis) 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 R-analysis-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 3.2×104-fold activity increase for 1-phenylbutylamine. 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. (Figure presented.).",
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Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone. / Kim, Hong Gon; Han, Sang Woo; shin, jong shik.

In: Advanced Synthesis and Catalysis, Vol. 361, No. 11, 06.06.2019, p. 2594-2606.

Research output: Contribution to journalArticle

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T1 - Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone

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