Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.
Understanding antibiotic resistance mechanisms is central to the development of anti‐infective therapies and genomics‐based drug discovery. Yet, many knowledge gaps remain regarding the resistance strategies employed against novel types of antibiotics from less‐explored producers such as anaerobic bacteria, among them the Clostridia. Through the use of genome editing and functional assays, we found that CtaZ confers self‐resistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum. Bioinformatics, biochemical analyses, and X‐ray crystallography revealed CtaZ as a founding member of a new group of GyrI‐like proteins. CtaZ is unique in binding a polythioamide scaffold in a ligand‐optimized hydrophobic pocket, thereby confining CTA. By genome mining using CtaZ as a handle, we discovered previously overlooked homologs encoded by diverse members of the phylum Firmicutes, including many pathogens. In addition to characterizing both a new role for a GyrI‐like domain in self‐resistance and unprecedented thioamide binding, this work aids in uncovering related drug‐resistance mechanisms.
Understanding antibiotic resistance mechanisms is central to the development of anti‐infective therapies and genomics‐based drug discovery. Yet, many knowledge gaps remain regarding the resistance strategies employed against novel types of antibiotics from less‐explored producers such as anaerobic bacteria, among them the Clostridia. Through the use of genome editing and functional assays, we found that CtaZ confers self‐resistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum. Bioinformatics, biochemical analyses, and X‐ray crystallography revealed CtaZ as a founding member of a new group of GyrI‐like proteins. CtaZ is unique in binding a polythioamide scaffold in a ligand‐optimized hydrophobic pocket, thereby confining CTA. By genome mining using CtaZ as a handle, we discovered previously overlooked homologs encoded by diverse members of the phylum Firmicutes, including many pathogens. In addition to characterizing both a new role for a GyrI‐like domain in self‐resistance and unprecedented thioamide binding, this work aids in uncovering related drug‐resistance mechanisms.
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