Inverting the Stereoselectivity of an NADH-Dependent Imine-Reductase Variant.

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.