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A reductive aminase switches to imine reductase mode for a bulky amine substrate

DOI: 10.1021/acscatal.2c06066 DOI Help

Authors: Amelia K. Gilio (University of York) , Thomas W. Thorpe (University of Manchester) , Alex Heyam (University of York) , Mark R. Petchey (University of York) , Balázs Pogrányi (University of York) , Scott P. France (Pfizer Worldwide Research and Development) , Roger M. Howard (Pfizer Worldwide Research and Development) , Michael J. Karmilowicz (Pfizer Worldwide Research and Development) , Russell Lewis (Pfizer Worldwide Research and Development) , Nicholas Turner (University of Manchester) , Gideon Grogan (University of York)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: January 2023
Diamond Proposal Number(s): 18598

Open Access Open Access

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 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, 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 equiv of three larger amines, in isolated yields of up to 93%.

Journal Keywords: biocatalysis; imine reductase; reductive aminase; chiral amine; NADPH

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-Macromolecular Crystallography

Added On: 16/01/2023 09:29


Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)