Engineering aromatic L-amino acid transaminase for the asymmetric synthesis of constrained analogs of L-phenylalanine

Byung Kwan Cho, Joo Hyun Seo, Taek Jin Kang, Juhan Kim, Hyung Yeon Park, Bon Su Lee, Byung Gee Kim

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

An enzymatic asymmetric synthesis was carried out for the preparation of enantiomerically pure L-diphenylalanine using the rationally engineered aromatic L-amino acid transaminase (eAroATEs) obtained from Enterobacter sp. BK2K-1. To rationally redesign the enzyme, structural model was constructed by the homology modeling. The structural model was experimentally validated by the site-directed mutagenesis of the predicted pyridoxal-5′-phosphate (PLP) binding site and the substrate-recognition region, and the cell-free protein synthesis of mutated enzymes. It was suggested that Arg281 and Arg375 were the key residues to recognize the distal carboxylate and α-carboxylate group of the substrates, respectively. The model also predicted that Tyr66 forms hydrogen bond with the phosphate moiety of PLP and interacts with the side chain attached to β-carbon of the amino acid substrate. Among the various site-directed mutants, Y66L variant was able to synthesize L-diphenylalanine with 23% conversion yield for 10 h, whereas the wild-type AroATEs was inactive for the transamination between diphenylpyruvate and L-phenylalanine as amino acceptor and amino donor, respectively.

Original languageEnglish
Pages (from-to)842-850
Number of pages9
JournalBiotechnology and Bioengineering
Volume94
Issue number5
DOIs
StatePublished - 5 Aug 2006

Keywords

  • Homology modeling
  • L-diphenylalanine
  • Site-directed mutagenesis
  • Transaminase
  • Unnatural amino acid

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