Inverting the stereoselectivity of an NADH-dependent imine reductase variant

Peter Stockinger; Niels Borlinghaus; Mahima Sharma; Benjamin Aberle; Gideon James Grogan; Juergen Pleiss; Bettina Nestl

doi:10.1002/cctc.202101057

Inverting the stereoselectivity of an NADH-dependent imine reductase variant

Peter Stockinger, Niels Borlinghaus, Mahima Sharma, Benjamin Aberle, Gideon James Grogan, Juergen Pleiss, Bettina Nestl

Chemistry

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Journal	ChemCatChem
Early online date	8 Oct 2021
DOIs	https://doi.org/10.1002/cctc.202101057
Publication status	E-pub ahead of print - 8 Oct 2021

Bibliographical note

Access to Document

10.1002/cctc.202101057Licence: CC BY

cctc.202101057
© 2021 The Authors.
Final published version, 1.64 MBLicence: CC BY

Cite this

@article{da7719ff72de42989a3677f939ab54ab,

title = "Inverting the stereoselectivity of an NADH-dependent imine reductase variant",

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.",

author = "Peter Stockinger and Niels Borlinghaus and Mahima Sharma and Benjamin Aberle and Grogan, {Gideon James} and Juergen Pleiss and Bettina Nestl",

note = "{\textcopyright} 2021 The Authors. ",

year = "2021",

month = oct,

day = "8",

doi = "10.1002/cctc.202101057",

language = "English",

journal = "ChemCatChem",

issn = "1867-3880",

publisher = "Wiley-Blackwell",

}

TY - JOUR

T1 - Inverting the stereoselectivity of an NADH-dependent imine reductase variant

AU - Stockinger, Peter

AU - Borlinghaus, Niels

AU - Sharma, Mahima

AU - Aberle, Benjamin

AU - Grogan, Gideon James

AU - Pleiss, Juergen

AU - Nestl, Bettina

PY - 2021/10/8

Y1 - 2021/10/8

N2 - 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.

AB - 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.

U2 - 10.1002/cctc.202101057

DO - 10.1002/cctc.202101057

M3 - Article

SN - 1867-3880

JO - ChemCatChem

JF - ChemCatChem

ER -