Structure, Activity and Stereoselectivity of NADPH-Dependent Oxidoreductases Catalysing theS-Selective Reduction of the Imine Substrate 2-Methylpyrroline

Abstract Oxidoreductases from Streptomyces sp. GF3546 [3546‐IRED], Bacillus cereus BAG3X2 ( Bc IRED) and Nocardiopsis halophila ( Nh IRED) each reduce prochiral 2‐methylpyrroline (2MPN) to ( S )‐2‐methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of Bc IRED and Nh IRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue—Tyr169—in 3546‐IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.