Enzyme‐Primed Native Chemical Ligation Produces Autoinducing Cyclopeptides in Clostridia

Abstract

Clostridia coordinate many important processes such as toxin production, infection, and survival by density‐dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone‐containing peptides, their true structures remain elusive. Here, we report the genome‐guided discovery of an AIP that controls endospore formation in Ruminiclostridium cellulolyticum. Through a combination of chemical synthesis and chemical complementation assays with a mutant strain, we reveal that the genuine chemical mediator is a homodetic cyclopeptide (cAIP). Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S→N acyl shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme‐primed, intramolecular NCL is a widespread feature of clostridial AIP biosynthesis.

Alternative Benzoxazole Assembly Discovered in Anaerobic Bacteria Provides Access to Privileged Heterocyclic Scaffold

“…Also, our discovery of a benzoxazole produced by a Clostridium sp. supports previous findings of anaerobes as a promising source of natural products and non‐canonical pathways [22b, 26] . Finally, we provide a first bioinformatic survey of putative benzoxazole BGCs that may guide future natural product discovery efforts.…”

Cleavage Off-Loading and Post-assembly-Line Conversions Yield Products with Unusual Termini during Biosynthesis

“…Alternatively, the β-sulfhydryl group of cysteinyl–S–T n might first form a thioester with the upstream acyl/peptidyl–S–T n –1 , followed by a rapid intramolecular S- to N-acyl transfer, similar to the process of native chemical ligation 22 used in peptide and protein synthesis and to the recently described mechanism observed during the biosynthesis of clostridial autoinducing peptides. 23 Therefore, with respect to the terminal thiocarboxylic formation, we surmise that (1) the PxbG Cy domains catalyzing thioesterification is the first step in heterocyclization as the energy barrier for conversion of thioester to amide bonds is much lower than that of amide to thioester bonds 24 and (2) due to the catalytic inefficiency of the PxbG Cy 3 and Cy 4 domains in heterocyclization relative to condensation, the thioester intermediates C1, F1, and G1 might be kinetically stable without being heterocyclized ( Figure 5 ). In particular, the Cy 4 domain is supposed to be the least efficient due to it only catalyzing thioester formation, albeit sharing ∼60% sequence similarities with Pxb Cy 1–3 ( Figure S3 ).…”

Rearrangement of Thiodepsipeptides by S → N Acyl Shift Delivers Homodetic Autoinducing Peptides

“…Finally, the discovery of this mechanism in the bacterial synthesis of homodetic peptides is independently corroborated by Hertweck and coworkers, who show that hexameric thiolactones produced in clostridia undergo the same transformation by S → N acyl shift chemistry. 21 This research provides fundamental insight into bacterial peptide biosynthesis and has implications for the future investigation of QS modulation and development of inhibitors of QS in several bacteria.…”

“…Our data also raises the question of whether other bacteria, such as L. plantarum and C. perfringens , rely on thiolactones and/or homodetic peptides as AIPs. Finally, the discovery of this mechanism in the bacterial synthesis of homodetic peptides is independently corroborated by Hertweck and co-workers, who show that hexameric thiolactones produced in clostridia undergo the same transformation by S → N acyl shift chemistry …”