A strategic Ser/Cys exchange in the catalytic triad unlocks an acyltransferase-mediated synthesis of thioesters and tertiary amides

Abstract

Expanding the toolbox of enzymatic reactions accessible to organic chemists is one of the major goals in biocatalysis. Here we describe the development of an acyltransferase variant from Mycobacterium smegmatis in which a strategic Ser/Cys exchange in the catalytic triad dramatically expanded its synthetic capability to yield a biocatalyst able to efficiently catalyse the formation of thioesters and tertiary amides in water. Preparative scale (250 mM) biotransformations were performed starting from different thiols and secondary amines with excellent yields and reactions times, using vinyl esters as acylating agents. The high substrate-to-catalyst ratio and the cofactor independence make this process a sustainable and cost-effective procedure that was successfully applied to the synthesis of acetyl coenzyme A as well as structurally simpler analogues. Computational studies provided insights into the enzymatic selectivity and substrate recognition. Preparative methods for thioester and tertiary amide formation are currently missing in the biocatalysis repertoire. Now, a mutation of serine to cysteine in the catalytic triad of an acyltransferase expands its synthetic capability to generate these compounds while retaining its natural activity towards alcohols.