Engineering the Active Site of an (S)‐Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

Henrik Land,Anna Szekrenyi,Federica Ruggieri,Per Berglund,Wolf‐Dieter Fessner

doi:10.1002/adsc.201901252

Abstract

AbstractA protein engineering approach for expanding the substrate scope of the (S)‐selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl‐ and benzyl‐substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2‐diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)‐1,2‐diphenylethylamine with ee>99% under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20‐30% (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)‐selective amine transaminase showing activity towards benzyl‐substituted primary amines.magnified image

Highlights

Chiral amines represent an important motif in many groups of fine chemicals, including pharmaceuticals[1] and agrochemicals.[2]
The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)-selective amine transaminase showing activity towards benzylsubstituted primary amines
The active site of Cv-amine transaminases (ATA) has been the subject of several engineering studies.[15b,c,18] With that in mind, Cv-ATA was chosen as a starting point for rational design driven expansion of the active site S pocket

Summary

Introduction

Chiral amines represent an important motif in many groups of fine chemicals, including pharmaceuticals[1] and agrochemicals.[2]. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups.

Results

Conclusion