Stockinger, Peter, Borlinghaus, Niels, Sharma, Mahima orcid.org/0000-0003-3960-2212 et al. (4 more authors) (2021) Inverting the stereoselectivity of an NADH-dependent imine reductase variant. ChemCatChem. ISSN 1867-3899
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-IREDMs variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 The Authors. |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Chemistry (York) |
Funding Information: | Funder Grant number BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL) BB/T017805/1 |
Depositing User: | Pure (York) |
Date Deposited: | 01 Nov 2021 15:20 |
Last Modified: | 07 Jan 2025 12:20 |
Published Version: | https://doi.org/10.1002/cctc.202101057 |
Status: | Published online |
Refereed: | Yes |
Identification Number: | 10.1002/cctc.202101057 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:179858 |