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

Balty, Clemence and Guillot, Alain and Fradale, Laura and Brewee, Clemence and Lefranc, Benjamin and Herrero, Christian and Sandström, Corine and Leprince, Jerome and Berteau, Olivier and Benjdia, Alhosna (2020). Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes. Journal of Biological Chemistry. 295 , 16665-16677
[Research article]

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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 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.

Authors/Creators:Balty, Clemence and Guillot, Alain and Fradale, Laura and Brewee, Clemence and Lefranc, Benjamin and Herrero, Christian and Sandström, Corine and Leprince, Jerome and Berteau, Olivier and Benjdia, Alhosna
Title:Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes
Series Name/Journal:Journal of Biological Chemistry
Year of publishing :2020
Volume:295
Page range:16665-16677
Number of Pages:13
Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
ISSN:0021-9258
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 3 Medical and Health Sciences > 301 Basic Medicine > Cell and Molecular Biology
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
URN:NBN:urn:nbn:se:slu:epsilon-p-109508
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-109508
Additional ID:
Type of IDID
DOI10.1074/jbc.RA120.015371
Web of Science (WoS)000598518600016
ID Code:21221
Faculty:NJ - Fakulteten för naturresurser och jordbruksvetenskap
Department:(NL, NJ) > Department of Molecular Sciences
Deposited By: SLUpub Connector
Deposited On:14 Jan 2021 12:23
Metadata Last Modified:15 Jan 2021 19:17

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