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