Redesigning the substrate specificity of ω-aminotransferase for the kinetic resolution of aliphatic chiral amines

Substrate specificity of the v-aminotransferase obtained from Vibrio fluvialis (v-ATVf) was rationally redesigned for the kinetic resolution of aliphatic chiral amines. v-ATVf 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 v-ATVf using homology modeling and the substrate structure- activity relationship. The homology model and the substrate structure-activity relationship showed that the active site of v-ATVf 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 a-carboxylate group. In the large substrate-binding site, R256 was predicted to recognize the a-carboxylate group of substrate thus obeying the dual substrate recognition mechanism of aminotransferase sub- group II enzymes. Among the several amino acid residues in the large substrate-binding site, W57 and W147, with their bulkysidechains,wereexpected torestricttherecognitionof 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.