Abstract
Substrate specificity of the ω‐aminotransferase obtained from Vibrio fluvialis (ω‐AT__Vf__) was rationally redesigned for the kinetic resolution of aliphatic chiral amines. ω‐AT__Vf__ showed unique substrate specificity toward aromatic amines with a high enantioselectivity (E > 100) for (S)‐enantiomers. However, the substrate specificity of this enzyme was much narrower toward aliphatic amines. To overcome the narrow substrate specificity toward aliphatic amines, we redesigned the substrate specificity of ω‐AT__Vf__ using homology modeling and the substrate structure‐ activity relationship. The homology model and the substrate structure‐activity relationship showed that the active site of ω‐AT__Vf__ consists of one large substrate‐binding site and another small substrate‐binding site. The key determinant in the small substrate‐binding site was D25, whose role was expected to mask R415 and to generate the electrostatic repulsion with the substrate's α‐carboxylate group. In the large substrate‐binding site, R256 was predicted to recognize the α‐carboxylate group of substrate thus obeying the dual substrate recognition mechanism of aminotransferase subgroup II enzymes. Among the several amino acid residues in the large substrate‐binding site, W57 and W147, with their bulky side chains, were expected to restrict the recognition of aliphatic amines. Two mutant enzymes, W57G and W147G, showed significant changes in their substrate specificity such that they catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity. Biotechnol. Bioeng. 2008;99: 275–284. © 2007 Wiley Periodicals, Inc.