Structure Based Semi-rational Engineering of OYE-family Enzymes for Non-natural Catalytic Activity

Abstract

The design of stereoselective catalysts remains a topic of great interest and importance to both chemists and the biomedical community at large. Recent work has established the development of enzymes as exceptionally stereoselective biocatalysts, particularly under the influence of visible light. Furthermore, both structural understanding and computational design have the potential to drive enzyme engineering efforts, particularly those which require high stereoselectivity. This project draws on structural determination and computational manipulation of proteins to aid in the engineering of C-C bond forming mutants. The crystal structures of one small molecule and six enzyme variants from four distinct proteins were solved using x-ray crystallography, producing the first reported high-resolution structures for three of these proteins: GluER, CSAR, and FlOYE. Substrate docking was used to rationalize differences in observed stereoselectivity between OYE-family members and was further used to select a set of candidate residues in the active site of enzyme OYE1 for directed evolution. Mutagenesis performed at these sites resulted in a biocatalyst with 20% greater yield and 10% better selectivity than the previous best variant. These findings support the idea that minor structural differences near the active site of an enzyme can have a profound impact on catalytic efficiency and stereoselectivity. Furthermore, the success of these experiments bodes well for future structural determination of OYE-family enzymes and substrate-docking-aided semi-rational directed evolution in a wide variety of systems.