Sandström, Corine
- Department of Molecular Sciences, Swedish University of Agricultural Sciences
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
SLU Authors
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