YcaO domains utilize ATP to activate amide backbones during peptide cyclodehydrations

Thiazole/oxazole-modified microcins (TOMMs) encompass a recently defined class of ribosomally synthesized natural products with a diverse set of biological activities. Although TOMM biosynthesis has been investigated for over a decade, the mechanism of heterocycle formation by the synthetase enzymes remains poorly understood. Using substrate analogs and isotopic labeling, we demonstrate that adenosine 5′-triphosphate (ATP) is utilized to directly phosphorylate the peptide amide backbone during TOMM heterocycle formation. Moreover, we present the first experimental evidence that the D-protein component of the heterocycle-forming synthetase (YcaO/DUF181 family member), formerly annotated as a docking/scaffolding protein involved in complex formation and regulation, is able to perform the ATP-dependent cyclodehydration reaction in the absence of the other TOMM biosynthetic proteins. Together, these data provide a greater level of detail into the biosynthesis of azol(in)e heterocycles in ribosomal natural products and prompt a reclassification of the enzymes involved in their installation.