Active-site engineering of ω-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

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

ω-Transaminase (ω-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 ω-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 ω-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 α-keto acids and α-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 α-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.

Original languageEnglish
Pages (from-to)6994-7002
Number of pages9
JournalApplied and Environmental Microbiology
Volume81
Issue number20
DOIs
Publication statusPublished - 2015 Jan 1

Fingerprint

transaminases
Transaminases
active sites
Alanine
alanine
Keto Acids
keto acids
Catalytic Domain
engineering
amino acid
Amino Acids
amino acids
acid
Ochrobactrum anthropi
mutation
Amination
Mutation
substitution
acids
mutagenesis

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Cite this

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title = "Active-site engineering of ω-transaminase for production of unnatural amino acids carrying a side chain bulkier than an ethyl substituent",
abstract = "ω-Transaminase (ω-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 ω-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 ω-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 α-keto acids and α-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 α-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.",
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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, Sang Woo; Park, Eul Soo; Dong, Joo Young; shin, jong shik.

In: Applied and Environmental Microbiology, Vol. 81, No. 20, 01.01.2015, p. 6994-7002.

Research output: Contribution to journalArticle

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