Dataset Information

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 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 E1 ubiquitin-activating and a YcaO superfamily member. Recent studies have demonstrated that the YcaO domain is responsible for cyclodehydration, while the TOMM E1 homologue is responsible for peptide recognition during azoline formation. Although all characterized TOMM biosynthetic clusters contain this canonical TOMM E1 homologue (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.

SUBMITTER: Dunbar KL

PROVIDER: S-EPMC4481143 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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

Dunbar Kyle L KL Tietz Jonathan I JI Cox Courtney L CL Burkhart Brandon J BJ Mitchell Douglas A DA

Journal of the American Chemical Society 20150612 24

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 E1 ubiquitin-activating and a YcaO superfamily member. Recent studies have demonstrated that the YcaO domain is responsible for cyclodehydration, while the TOMM E1 homologue is responsible for peptide recognition during azoline formation. ...[more]

PMID: 26024319

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Project description:Ribosomally synthesized and Post-translationally modified Peptides (RiPPs) take advantage of the ribosomal translation machinery to generate linear peptides that are subsequently modified with heterocycles and/or macrocycles to impose three-dimensional structure and thwart degradation by proteases. Although RiPP precursors are limited to proteinogenic amino acids, post-translational modifications (PTMs) can alter the structure of individual amino acids and thereby improve the stability and biological activity of the molecule. These "tailoring modifications" often occur on amino acid side chains-for example, hydroxylation, methylation, halogenation, prenylation, and acylation-but can also take place within the backbone, as in epimerization, or can result in capping of the N- or C-terminus. At one extreme, these modifications can be essential to the activity of the RiPP, either as a compulsory step in reaching the final molecule or by imparting chemical functionality required for biological activity. At the other extreme, tailoring PTMs may have little effect on the activity in an in vitro setting-possibly because of test conditions that do not match the biological context in which the PTMs evolved. Establishing the molecular basis for the function of tailoring PTMs often requires a three-dimensional structure of the RiPP bound to its biological target. These structures have revealed roles for tailoring PTMs that include providing additional hydrogen bonds to targets, rigidifying the RiPP structure to reduce the entropic cost of binding, or altering the secondary structure of the peptide backbone. Bacterial RiPPs are particularly suited to structural characterization, as they are relatively easy to isolate from laboratory cultures or to produce in a heterologous host. The identification of new tailoring PTMs within bacteria is also facilitated by clustering of the genes encoding tailoring enzymes with those of the RiPP precursor and primary modification enzymes. In this Account, we describe the effects of tailoring PTMs on RiPP structure, their interactions with biological targets, and their influence on RiPP stability, with a focus on bacterial RiPP classes. We also discuss the enzymes that generate tailoring PTMs and highlight examples of and prospects for engineering of RiPPs.

Dataset Information

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

Publications

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

Similar Datasets

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