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

Abstract

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.

Features and technical applications of ω-transaminases

“…Error-prone PCR was used to generate a mutant library, which was enriched to select mutant enzymes that successfully resolved racemic 2-aminoheptane. Redesigning substrate specificity of the V. fluvialis 5-TA using homology modeling and site saturated mutagenesis was also reported by the same group (Cho et al 2008).…”

Process considerations for the asymmetric synthesis of chiral amines using transaminases

“…For instance Martin et al (2007) managed to improve the activity of a transaminase by a factor of almost 300, while at the same time improving the stability of the enzyme toward the process conditions, yielding a much more economic process. Other examples are given in reports by Rothman et al (2004) and Yun et al (2005) who managed to overcome product inhibition by directed evolution, Cho et al (2008) redesigned the substrate specificity of an v-transaminase for the kinetic resolution of aliphatic chiral amines.…”

Transaminases and their Applications

“…This might be caused by steric hindrances of two tryptophans in the larger binding site of the transaminase. Changing the amino acid from tryptophan to glycine reduces the hydrophobic interaction and allows binding of aromatic groups or aliphatic chains [119].…”