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

Abstract

Thiazole/oxazole-modified microcins (TOMMs) are a class of posttranslationally 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 E1 ubiquitin-activating and a YcaO superfamily member. Recent studies have demonstrated that the YcaO domain is responsible for cyclodehydration while the TOMM E1 homolog is responsible for peptide recognition during azoline formation. Although all characterized TOMM biosynthetic clusters contain this canonical TOMM E1 homolog (C domain), we also identified a second, highly divergent E1 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.

In Vitro Biosynthesis and Substrate Tolerance of the Plantazolicin Family of Natural Products

“…This result is in stark contrast to several previously studied TOMM synthetases, where the C-protein, which contains the ~90 residue RRE, was sufficient for effective peptide binding (12, 46). We hypothesize that some C-proteins must leverage their association with other synthetase components to induce structural rearrangements that potentiate leader peptide binding (49). …”

“…To assess the interaction of non-fluorescent peptides with the PZN synthetase, a competition FP binding assay was used as previously described (49). MBP-tagged precursor peptides were serially diluted and mixed with 25 nM of a fluorescent peptide and synthetase component(s).…”

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…Mutational studies using the sactipeptide RiPP biosynthetic machinery has also been performed, revealing the importance of the RRE for sactipeptide maturation (Wieckowski et al, 2015). Interestingly, the RRE domain sometimes may exist as a stand-alone protein and not as a subdomain within a RiPP biosynthetic enzyme as recently observed for lasso peptides and thiazole/oxazole-modified peptide gene clusters (Burkhart et al, 2015, Dunbar et al, 2015, Elsayed et al, 2015, Gavrish et al, 2014, Inokoshi et al, 2012, Li et al, 2015, Metelev et al, 2015). …”

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

“…188, 268 In public genome databases, this protein is most often annotated as “Ocin-ThiF-like.” Importantly, this ThiF-like partner protein contains the RRE, which binds the precursor peptide, and potentiates YcaO catalyzed cyclodehydration. 27, 45, 185 Accordingly, the thiopeptide cyclodehydratase is most accurately described by the combined activities of the partner protein and YcaO because both are necessary for azoline biosynthesis. 45 Notwithstanding these observations, there are rare examples of thiopeptide producers that do not locally encode a ThiF-like protein homolog (e.g.…”