Inverting the Stereoselectivity of an NADH‐Dependent Imine‐Reductase Variant

Stockinger, Peter; Borlinghaus, Niels; Sharma, Mahima; Aberle, Benjamin; Grogan, Gideon; Pleiss, Jürgen; Nestl, Bettina M.

doi:10.1002/cctc.202101057

Cited by 12 publications

(10 citation statements)

References 43 publications

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“…A comparison of the monomer with structures in the PDB using the DALI server 24 revealed closest structural similarity with the IREDs from Myxococcus stipitatus (6TOE; 51% sequence identity; rmsd 1.3 Å over 291 Cα atoms) 25 and Streptosporangum roseum (5OCM; 34%; 1.8 Å over 290). 26 Following building of the protein atoms and water molecules, clear electron density was observed within each active site that could in each case be modeled as NADP + .…”

Section: Resultsmentioning

confidence: 96%

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate

et al. 2023

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Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzymecatalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structureguided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.

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Section: Resultsmentioning

confidence: 96%

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate

et al. 2023

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Section: Imine Reductases and Reductive Aminases (Ireds And Redams)mentioning

confidence: 99%

“…Beneficial mutations were recombined, and the final variant containing five mutations (A241V/H242Y/N243D/V244Y/ A245L) exhibited a switch of enantio-selectivity from (R)configuration (99% ee) to (S)-configuration with 91% ee. 108 The IREDs toolbox was further expanded by mining and characterizing novel IREDs using tools such as BLAST. Several panels of bacterial IREDs have been identified to catalyze the formation of primary and secondary amines.…”

Section: Discovery History Of Evolution and Substrate Scope Of Iredsmentioning

confidence: 99%

Biocatalytic reductive aminations with NAD(P)H-dependent enzymes: enzyme discovery, engineering and synthetic applications

Yuan,

Yang,

et al. 2024

Chem. Soc. Rev.

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“…In 2021, Pleiss and Nestl et al. inverted the stereoselectivity of IRED from Myxococcus stipitatus (NADH‐IRED‐Ms) [22] . After screening site‐saturation mutagenesis libraries, the variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereoselectivity in 2‐MPN reduction compared to the wild type.…”

Section: Different Catalytic Mechanisms For Asymmetric Reduction Of C...mentioning

confidence: 99%

Imine Reductases: Multifunctional Biocatalysts with Varying Active Sites and Catalytic Mechanisms

Huang

Shao

2022

ChemCatChem

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The synthesis of chiral amines is significant in the pharmaceutical industry. Imine reductase (IRED), a promising biocatalyst that was previously known to only catalyze asymmetric imine reduction (IR), was revealed to achieve direct asymmetric reductive amination (RA) of ketones with excess amines, producing secondary and tertiary amines. Moreover, conjugate reduction (CR) and RA activity by a single IRED has been reported for the preparation of valuable amine diastereomers. IREDs catalyzing these different reactions share the same standard quaternary structure, but possess varying active sites, indicating correlations and differences between their catalytic mechanisms. In this review, we trace the catalytic mechanisms reported for IRED‐catalyzed IR, RA, and CR‐RA. Insights obtained from structural and protein engineering in understanding the IRED‐catalyzed asymmetric synthesis are also included. This review will help readers acquire comprehensive insights into the catalytic mechanism of IREDs and ultimately inspire the engineering of IREDs for industrial applications.

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Inverting the Stereoselectivity of an NADH‐Dependent Imine‐Reductase Variant

Cited by 12 publications

References 43 publications

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate

Biocatalytic reductive aminations with NAD(P)H-dependent enzymes: enzyme discovery, engineering and synthetic applications

Imine Reductases: Multifunctional Biocatalysts with Varying Active Sites and Catalytic Mechanisms

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