Engineering aromatic L-amino acid transaminase for the asymmetric synthesis of constrained analogs of L-phenylalanine.
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 alpha-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 beta-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.
Cho
Byung-Kwan
BK
School of Chemical and Biological Engineering and Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea.
Seo
Joo-Hyun
JH
Kang
Taek-Jin
TJ
Kim
Juhan
J
Park
Hyung-Yeon
HY
Lee
Bon-Su
BS
Kim
Byung-Gee
BG