Inverting the Stereoselectivity of an NADH‐Dependent Imine‐Reductase Variant

Abstract

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (<i>R</i>)-selective IRED from <i>Myxococcus stipitatus</i> (NADH-IRED-<i>Ms</i>) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (<i>S</i>)-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-<i>Ms</i>. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2-methylpyrroline compared to the wild-type and afforded the (<i>S</i>)-amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal-structures of the NADPH-dependent (<i>R</i>)-IRED-<i>Ms</i> wild-type enzyme and the NADH-dependent NADH-IRED-<i>Ms</i> variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.