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. © 2006 Wiley Periodicals, Inc.