Identification of an Auxiliary Leader Peptide-Binding Protein Required for Azoline Formation in Ribosomal Natural Products.

Thiazole/oxazole-modified microcins (TOMMs) are a class of post-translationally modified peptide natural products bearing azole and azoline heterocycles. The first step in heterocycle formation is carried out by a two component cyclodehydratase comprised of an El ubiquitin- activating and a YcaO superfamily member. Recent studies have demonstrated that the YcaO domain is responsible for cyclodehydration, while the TOMM El homologue is responsible for peptide recognition during azoline formation. Although all characterized TOMM biosynthetic clusters contain this canonical TOMM El homologue (C domain), we also identified a second, highly divergent El superfamily member, annotated as an Ocin-ThiF-like protein (F protein), associated with more than 300 TOMM biosynthetic clusters. Here we describe the in vitro reconstitution of a novel TOMM cyclodehydratase from such a cluster and demonstrate that this auxiliary protein is required for cyclodehydration. Using a combination of biophysical techniques, we demonstrate that the F protein, rather than the C domain, is responsible for engaging the peptide substrate. The C domain instead appears to serve as a scaffolding protein, bringing the catalytic YcaO domain and the peptide binding Ocin-ThiF-like protein into proximity. Our findings provide an updated biosynthetic framework that provides a foundation for the characterization and reconstitution of approximately 25% of bioinformatically identifiable TOMM synthetases. [ABSTRACT FROM AUTHOR]