Design and evolution of an enzyme with a non-canonical organocatalytic mechanism

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions^1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis⁵, here we report the generation of a hydrolytic enzyme that uses N_δ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design^6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free N_δ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. N_δ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine¹¹, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.