Grogan, Gideon James orcid.org/0000-0003-1383-7056, Gilio, Amelia, Thorpe, Thomas et al. (8 more authors) (2023) A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate. ACS Catalysis. 1669–1677. ISSN 2155-5435
Abstract
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 enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines are 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, structure-guided 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 equivalents of three larger amines, in isolated yields of up to 93%.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2023 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 Feb 2023 11:00 |
Last Modified: | 07 Jan 2025 12:10 |
Published Version: | https://doi.org/10.1021/acscatal.2c06066 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1021/acscatal.2c06066 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:195929 |
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Description: A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate
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