Switching the Cofactor Specificity of an Imine Reductase

Abstract In the last years, imine reductases (IREDs) have gained importance for the formation of chiral amines by catalyzing asymmetric reductions of imines and chemo‐ and stereoselective reductive aminations. However, all characterized members of this steadily growing enzyme family demonstrated strict preference for NADPH, which entails reduced possibilities for efficient cofactor regenerations and limitations in the construction of whole cell systems. To alter the cofactor specificity from NADPH to NADH, we applied the “Cofactor Specificity Reversal—Structural Analysis and Library Design” (CSR‐SALAD) mutagenesis strategy and enlarged the mutant library by further amino acid replacements. This engineering approach has been shown to result in IRED variants with up to 2900‐fold improved NADH/NADPH specificity and completely recovered activity in the reduction of 2‐methyl pyrroline (2MP).