Mutagenesis of a Single Site Inverts the Stereopreference of Imine Reductase

Development of a generally applicable means to invert the stereoselectivity of an enzymatic reaction is of paramount significance. Through protein structure-guided mutagenesis, Met235 was identified as a crucial residue influencing the stereoselectivity of the imine reductase AtIRED-catalyzed reduction of sterically demanding 1-substituted dihydro-β-carbolines (DHβCs), particularly with single mutants M235A/C/G/I/S/T/V displaying simultaneously inverted stereoselectivity and improved catalytic activity relative to the wild-type (WT) enzyme. Using the best variant M235A as the biocatalyst, five 1-substituted tetrahydro-β-carbolines (THβCs) of (R)-configuration were afforded in 48–81% isolated yields with 89 → 99% ee. Combined with our previous synthesis of the (S)-stereoisomer using WT and other variants, we have established stereocomplementary access to these THβCs. Based on the solved crystal structure of variant M235A complexed with NADP+ and substrate 1-t-butyl-DHβC, the M235A mutation-induced relief of undesired steric clashes was rationalized as the main cause of the observed stereoselectivity inversion and activity enhancement. This influence on stereopreference exerted by the single M235A mutation was transferred successfully to Y-type IREDs and, in part, to D-type IREDs, representing the first demonstration of this kind of knowledge transfer between imine reductases. The current study identifies a stereocontrol element of IREDs, and it offers a potentially generic strategy to switch the stereopreference of these fascinating enzymes.