Inverting the stereoselectivity of an NADH‐dependent imine‐reductase variant

DOI: 10.1002/cctc.202101057

Authors: Peter Stockinger (University of Stuttgart) , Niels Borlinghaus (University of Stuttgart) , Mahima Sharma (University of York) , Benjamin Aberle (University of Stuttgart) , Gideon Grogan (University of York) , Jürgen Pleiss (University of Stuttgart) , Bettina M. Nestl (University of Stuttgart)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chemcatchem

State: Published (Approved)
Published: October 2021
Diamond Proposal Number(s): 9948

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 (( R )-IRED- Ms _V8) 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 ( R )-IRED- Ms _V8. 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 ( R )-IRED- Ms _V8 variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.

Journal Keywords: biocatalysis; imine reductase; stereoselectivity; molecular dynamics simulations; crystal structure

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 11/10/2021 08:49

Documents:
cctc.202101057.pdf

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)