In Vitro Biosynthesis of the Core Scaffold of the Thiopeptide Thiomuracin

Thiopeptides are potent antibiotics that inhibit protein synthesis. They are made by a remarkable post-translational modification process that transforms a linear peptide into a polycyclic structure. We present here the in vitro biosynthesis of the core scaffold of thio­muracin catalyzed by six proteins. We show that cyclo­dehydration precedes dehydration, and that dehydration is catalyzed by two proteins in a tRNAGlu-dependent manner. The enzyme that generates the pyridine core from two dehydro­alanines ejects the leader peptide as a C-terminal carboxamide. Mutagenesis studies of the enzyme TbtD identified important residues for a formal [4+2] cycloaddition process. The core structure of thio­muracin exhibits similar antimicrobial activity to other known congeners, illustrating that in vitro biosynthesis is a viable route to potent antibiotics that can be explored for the rapid and renewable generation of analogues.