Structural and Biochemical Characterization of the ATP-Binding YcaO Domain

Thiazole/oxazole-modified microcins (TOMMs) are a family of RiPP natural products that contain a heterocycle formed between the peptide backbone and an amino acid side chain. Despite intensive research, the cyclodehydratase responsible for azoline biogenesis in TOMMs remains enigmatic. The collaboration of two proteins, C and D, is required for cyclodehydration. The C protein is homologous to E1 enzymes, while the D protein is within the YcaO superfamily. Recent studies have demonstrated that TOMM YcaOs phosphorylate amide carbonyl oxygens to facilitate azoline formation. Here we report the X-ray crystal structure of an uncharacterized YcaO from Escherichia coli (Ec-YcaO). Ec-YcaO harbors an unprecedented fold and ATP-binding motif that is conserved among TOMM YcaOs and is required for cyclodehydration. This study identifies the YcaO active site and paves the way for the characterization of the numerous YcaO domains not associated with TOMM biosynthesis.