Protein Engineering of Nicotinamide-Dependent Cyclohexanone Reductase Towards Non-Native Photocatalytic Oxime Cyclization

Abstract

Drug candidates are currently dominated by relatively flat structures due to the synthetic challenge of constructing tetrasubstituted centers that would allow for the expanded sampling of drug-like chemical spaces. Achieving this stereoselectively is critical for the pharmaceutical industry as opposing enantiomers often have differing biological activity. Enzymes offer the potential to catalyze reactions with excellent efficacy and enantioselectivity. Recently, biocatalysts have been adapted to perform reactions outside of their native reactivities, making them even more valuable for asymmetric syntheses. By exploiting the promiscuous reactivity of flavin, a non-natural reactivity of the NCR ene-reductase was discovered to catalyze a Csp3-Csp3 bond formation via a photoinduced radical cyclization, producing a -lactam, a common biologically active motif, with the installation of a chiral -tertiary amine center. Although the wild type enzyme demonstrated moderate reactivity, reporting 32% substrate conversion with 85:15 er, optimization of reaction conditions and construction of 18 site-saturated mutant libraries enabled screenings that lead to mutants bearing beneficial amino acid substitutions. One particular variant (Y343W) was shown to have remarkable improvements of 97% yield and 96:4 er, with a suggestive selective conformer consumption, leading to the hypothesis that the particular mutation may contribute to the stabilization of specific substrate orientations.