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

Abstract

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 ( R )‐selective IRED from Myxococcus stipitatus (NADH‐IRED‐ Ms ) yielding a NADH‐dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and ( S )‐selectivity in asymmetric reductions has yet been reported. Herein, we applied semi‐rational enzyme engineering to switch the selectivity of NADH‐IRED‐ Ms . 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 ( S )‐amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal‐structures of the NADPH‐dependent ( R )‐IRED‐ Ms wild‐type enzyme and the NADH‐dependent NADH‐IRED‐ Ms variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.