Stereodivergent evolution of KpADH for the asymmetric reduction of diaryl ketones with para-substituents

Abstract

KpADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcohols. Substrate specificity toward diaryl ketone substrates with different substituents at phenyl group was investigated. The results reveal that the para-position of diaryl ketones plays important role in manipulating the stereoselectivity of KpADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and saturation mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of KpADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% (S) and 99% (R) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone (5a), and e.e. values of >99% (S) and 98% (R) toward (4-methylphenyl)-2-pyridinylmethanone (6a), respectively. Kinetic characterization and interaction analysis further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para-substituents and also provides guidance for the engineering of homologous alcohol dehydrogenases toward bulky-bulky ketones.