8A5Z

Imine Reductase from Ensifer adhaerens A208N mutant in complex with NADP+

Summary for 8A5Z

• Entry DOI: 10.2210/pdb8a5z/pdb
• Related: 8A3X
• Descriptor: NAD_binding_2 domain-containing protein, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, IODIDE ION, ... (4 entities in total)
• Functional Keywords: amine, nadp, imine reductase oxidoreductase, oxidoreductase
• Biological source: Ensifer adhaerens
• Total number of polymer chains: 2
• Total formula weight: 64798.53

Authors

Gilio, A.K.,Grogan, G. (deposition date: 2022-06-16, release date: 2023-03-08, Last modification date: 2024-02-07)

Primary citation

Gilio, A.K.,Thorpe, T.W.,Heyam, A.,Petchey, M.R.,Pogranyi, B.,France, S.P.,Howard, R.M.,Karmilowicz, M.J.,Lewis, R.,Turner, N.,Grogan, G.
A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate.
Acs Catalysis, 13:1669-1677, 2023

PubMed 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 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()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%.

PubMed: 36776386
DOI: 10.1021/acscatal.2c06066

Experimental method

X-RAY DIFFRACTION (2.31 Å)