Premium
Engineering aromatic L ‐amino acid transaminase for the asymmetric synthesis of constrained analogs of L ‐phenylalanine
Author(s) -
Cho ByungKwan,
Seo JooHyun,
Kang TaekJin,
Kim Juhan,
Park HyungYeon,
Lee BonSu,
Kim ByungGee
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20902
Subject(s) - transamination , stereochemistry , chemistry , moiety , carboxylate , amino acid , active site , phenylalanine , pyridoxal phosphate , enzyme , transaminase , enantioselective synthesis , protein engineering , site directed mutagenesis , biochemistry , mutant , catalysis , cofactor , gene
An enzymatic asymmetric synthesis was carried out for the preparation of enantiomerically pure L ‐diphenylalanine using the rationally engineered aromatic L ‐amino acid transaminase (eAroAT Es ) 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 AroAT Es was inactive for the transamination between diphenylpyruvate and L ‐phenylalanine as amino acceptor and amino donor, respectively. © 2006 Wiley Periodicals, Inc.