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

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.
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https://hal.inrae.fr/hal-03332307
Contributor : Roselyne Tâche <>
Submitted on : Thursday, September 2, 2021 - 3:50:22 PM
Last modification on : Saturday, September 4, 2021 - 3:28:10 AM

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Clémence Balty, Alain Guillot, Laura Fradale, Clémence Brewee, Benjamin Lefranc, et al.. Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2020, 295 (49), pp.16665-16677. ⟨10.1074/jbc.RA120.015371⟩. ⟨hal-03332307⟩

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