Ruminococcin C, an anti-clostridial sactipeptide produced by a prominent member of the human microbiota Ruminococcus gnavus

• Published in: The Journal of biological chemistry Vol. 294; no. 40; pp. 14512 - 14525
• Main Authors: Balty, Clémence, Guillot, Alain, Fradale, Laura, Brewee, Clémence, Boulay, Mylène, Kubiak, Xavier, Benjdia, Alhosna, Berteau, Olivier
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04-10-2019; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence - genetics; antibiotics; antimicrobial peptide (AMP); Bacteriocins - biosynthesis; Bacteriocins - chemistry; Bacteriocins - genetics; Biochemistry, Molecular Biology; Clostridiales - enzymology; Clostridiales - genetics; Clostridium perfringens - chemistry; Clostridium perfringens - genetics; Clostridium perfringens - pathogenicity; enzyme; Enzymology; Gastrointestinal Microbiome - genetics; Humans; Life Sciences; microbiome; Multigene Family - genetics; natural product biosynthesis; peptide biosynthesis; Peptide Biosynthesis - genetics; Peptides - chemistry; Peptides - genetics; Protein Processing, Post-Translational - genetics; radical; radical AdoMet enzyme; radical SAM enzyme; Ribosomes - genetics; RiPP; ruminococcin; ruminococcin C; Sterile Alpha Motif - genetics; Sulfides - chemistry; Symbiosis - genetics; enzyme; natural product biosynthesis; RiPP; peptide biosynthesis; radical; radical SAM enzyme; ruminococcin; radical AdoMet enzyme; ruminococcin C; antibiotics; microbiome; antimicrobial peptide (AMP)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009416

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus. This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens. By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to–α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.