An enzymatic asymmetric synthesis was carried out for the preparation of enantiomerically pure L-cliphenylalanine using the rationally engineered aromatic L-amino acid transaminase (eAroATEs) obtained from Enterobactersp. 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 cliphenylpyruvate and L-phenylalanine as amino acceptor and amino donor, respectively. (c) 2006 Wiley Periodicals, Inc.