Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes |
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Authors: | Clé mence Balty,Alain Guillot,Laura Fradale,Clé mence Brewee,Benjamin Lefranc,Christian Herrero,Corine Sandströ m,Jé rô me Leprince,Olivier Berteau,Alhosna Benjdia |
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Affiliation: | 1.Micalis Institute, ChemSyBio, INRAE, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France;2.INSERM U1239, PRIMACEN, Université de Normandie-Rouen, Rouen, France;3.ICMMO, CNRS, Université Paris-Saclay, Orsay, France;4.Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden |
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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 Cα-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-α-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 Cα 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 α,β-dehydro-amino acid intermediate during Cα-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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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 |
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