Engineering the enantioselectivity of a novel imine reductase from Streptomyces viridochromogenes for the dynamic kinetic reductive amination of cyclic β-ketoester

Abstract

N-substituted β-amino esters as an important class of chiral amines, are widely used as building blocks in pharmaceuticals. A novel imine reductase SvIRED was identified through gene mining from Streptomyces viridochromogenes with high enantioselectivity of > 99 % ee (1R, 2S), 95 % de (cis:trans), towards 2-oxocyclohexane-1-carboxylate and cyclopropylamine. SvIRED exhibited the highest activity at pH 7.0 and 40 °C. A variety of amine donors could be used by SvIRED and produce chiral N-substituted β-amino esters with different enantioselectivity. Employing methylamine (i) and pyrrolidine (n) as amine donors, SvIRED exhibited reversed enantioselectivity of 23 % ee (1S, 2R) and 79 % ee (1S, 2R) respectively. Through alanine scanning, M183 and H244 were identified with remarkable roles in governing enantioselectivity towards i and n. Compared with WT SvIRED, the enantioselectivity of M183A was inverted to ee value of >99 % (1R, 2S) and de value of >99 % (cis:trans) towards i, and the enantioselectivity of H244A was inverted to ee value of 88 % (1R, 2S) and de value of >99 % (cis:trans) towards n. Interaction analysis indicated the orientation of ester group was vital for manipulation of enantioselectivity. This study provides novel Imine reductase mutants for enantioselective synthesis of N-substituted β-amino esters with different amine donors.