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Research article2020Peer reviewedOpen access

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes

Balty, Clemence; Guillot, Alain; Fradale, Laura; Brewee, Clemence; Lefranc, Benjamin; Herrero, Christian; Sandstrom, Corine; Leprince, Jerome; Berteau, Olivier; Benjdia, Alhosna

Abstract

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four C-alpha-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-alpha-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by C-alpha H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an alpha,beta-dehydro-amino acid intermediate during C-alpha-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

Keywords

radical SAM enzyme; radical AdoMet enzyme; antimicrobial peptide; microbiota; microbiome; antibiotics; enzyme; peptide biosynthesis; RiPP; ruminococcin C; RumC; sactipeptide; antimicrobial peptide (AMP); metalloenzyme; radical; enzyme catalysis

Published in

Journal of Biological Chemistry
2020, Volume: 295, number: 49, pages: 16665-16677 Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    UKÄ Subject classification

    Cell and Molecular Biology

    Publication identifier

    DOI: https://doi.org/10.1074/jbc.RA120.015371

    Permanent link to this page (URI)

    https://res.slu.se/id/publ/109508