Redesigning the substrate specificity of omega-aminotransferase for the kinetic resolution of aliphatic chiral amines.
Substrate specificity of the omega-aminotransferase obtained from Vibrio fluvialis (omega-ATVf) was rationally redesigned for the kinetic resolution of aliphatic chiral amines. omega-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 omega-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 omega-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 alpha-carboxylate group. In the large substrate-binding site, R256 was predicted to recognize the alpha-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.
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Korea.
Byung-Kwan
BK
Cho
Hyung-Yeon
HY
Park
Joo-Hyun
JH
Seo
Juhan
J
Kim
Taek-Jin
TJ
Kang
Bon-Su
BS
Lee
Byung-Gee
BG
Kim
D000588:Amines / Q000138:chemical synthesis*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry*
D000588:Amines; D000637:Transaminases