Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

Abstract

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.

An Unexpected Split‐Merge Pathway in the Assembly of the Symmetric Nonribosomal Peptide Antibiotic Closthioamide

“…Although the presence of CtaB and a PLP‐dependent decarboxylase (CtaF) in the genetic locus is consistent with such a pathway, we recently demonstrated that CtaF is involved in the assembly of the poly‐βAla backbone of CTA (Figure 1 C). [8a] Thus, we predicted that the diamine core of CTA would originate from a novel pathway involving CtaB and the AKR CtaK.…”

“…Based on the results described herein, we propose a model for the complete biosynthetic pathway to CTA (Scheme 1). The biosynthesis begins with the conversion of chorismate to PHBA by CtaA and the assembly of the amide backbone on CtaE (CtaE‐amido‐ 1 ) by CtaD and CtaF–G [3a, 8a] . Next, CtaE‐amido‐ 1 , is iteratively thioamidated by CtaC to afford CtaE‐ 1 [8b] .…”

Mining and unearthing hidden biosynthetic potential

“…Its biosynthesis, however, has remained enigmatic until recently. Now it was demonstrated that CTA is biosynthesized via a novel thiotemplated peptide assembly line that differs from canonical NRPSs and therefore escaped previous genomic analyses with automated software tools 100,101 . This example showcases the limitations of automated genome mining efforts relying on characterized biosynthetic mechanisms.…”

Ribosome-independent peptide biosynthesis: the challenge of a unifying nomenclature

“…In many biosynthetic pathways, however, only a portion of the biosynthetic enzymes are encoded as standalone proteins. Although a pure type II NRPS would require that all biosynthetic enzymes were standalone proteins, such examples are exceedingly rare, 20,21 and type II systems predominantly occur as hybrids with type I NRPS or PKS assembly lines. The standalone portion of the biosynthetic enzymes are most commonly A domains, CPs or A-CP didomains.…”