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Published on: February 24, 2018
Inverting the Stereoselectivity of an NADH-Dependent Imine-Reductase Variant
Peter Stockinger1, Niels Borlinghaus1, Mahima Sharma2
1Institute of Biochemistry and Technical Biochemistry Department of Technical Biochemistry Universitaet Stuttgart Allmandring 31 70569 Stuttgart Germany.
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.
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