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 post-translationally 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 non-natural 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 (RRE), present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the RRE and the core peptide for the installation of post-translational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

Radical SAM Enzymes and Ribosomally‐Synthesized and Post‐translationally Modified Peptides: A Growing Importance in the Microbiomes

“…For instance, they have been reported to be involved in key biological processes such as DNA repair ( Rebeil et al, 1998 ; Chandor et al, 2006 ; Friedel et al, 2006 ; Benjdia et al, 2012 ), protein posttranslational modification ( Ollagnier et al, 1996 ; Berteau et al, 2006 ; Benjdia et al, 2007a ; Benjdia et al, 2007b ; Benjdia et al, 2008 ; Arragain et al, 2009 ; Benjdia et al, 2009 ; Benjdia et al, 2010 ), nucleic acid modification ( Pierrel et al, 2003 ; Grove et al, 2011 ) and the biosynthesis of cofactors ( Layer et al, 2006 ; Decamps et al, 2012 ; Harmer et al, 2014 ; Philmus et al, 2015 ) and vitamins ( Vander Horn et al, 1993 ; Sanyal et al, 1994 ; Kriek et al, 2007 ; Chatterjee et al, 2008 ). More recently, there has been a tremendous increase in the discovery of radical SAM enzymes involved in RiPP biosynthetic pathways ( Wang and Frey, 2007 ; Fluhe et al, 2012 ; Pierre et al, 2012 ; Allen and Wang, 2014 ; Benjdia et al, 2015 ; Benjdia et al, 2017a ; Parent et al, 2018 ; Balty et al, 2019 ; Imai et al, 2019 ; Balty et al, 2020 ).…”

“…First dubbed sactibiotics ( Murphy et al, 2011 ), they are now collectively named sactipeptides ( Arnison et al, 2013 ). In addition to hyicin, a RiPP highly homologous to subtilosin A identified in Staphylococcus hyicus ( Duarte et al, 2018 ), ruminococcin C ( Balty et al, 2019 ; Balty et al, 2020 ), thuricin Z ( Hudson et al, 2019 ; Mo et al, 2019 ), and streptosactin ( Bushin et al, 2020 ) were recently discovered and their biosynthetic pathway deciphered, bringing the number of known sactipeptides to seven.…”

“…Indeed, bacteria can adopt complex lifestyles ( Hibbing et al, 2010 ), with RiPPs providing not only a competitive advantage against competing species ( Telhig et al, 2020 ), but also playing a major role in communication ( Ibrahim et al, 2007 ; Liu et al, 2010 ), biofilm formation and the acquisition of metals ( Hider and Kong, 2010 ; Dassama et al, 2017 ). More recently, RiPPs have also been proposed to play a pivotal role in the homeostasis of the human microbiota ( Benjdia and Berteau, 2016 ; Balskus, 2018 ; Chittim et al, 2018 ; Balty et al, 2020 ), with the recent discovery of several novel antibiotics from this complex ecosystem ( Rothschild et al, 2018 ) including colicin V ( Cohen et al, 2018 ), humimycin ( Chu et al, 2018 ) and ruminococcin C ( Balty et al, 2019 ; Chiumento et al, 2019 ; Balty et al, 2020 ).…”

Current Advancements in Sactipeptide Natural Products

“…Starting from precursor A, the first thioether bridges (i.e., the Cys24-Glu31 and Cys45-Arg53 bridges) are installed in each hairpin domain (intermediate B). Then the second thioether bridges, Cys22-Asn35 (intermediate C) and Cys41-Arg61, are formed, leading to the production of the mature peptide C2MC2 (species E) with two hairpin domains (Balty et al, 2019(Balty et al, , 2020. enzyme gene thnB, a protease thnP, three-component ABC transporter gene thnD/E/T, transcriptional regulator gene thnR, and an unknown function gene thnI (Figure 2).…”

“…This characteristic is unique within the currently reported sactipeptide family; The acceptor amino acids Ala12, Asn16, Arg34, and Lys42 of the sactionines in RumC1 are D-configurations which are opposite to a recent report by Berteau and coworkers. The compact double hairpin-like structure gives RumC1 a high tolerance with pH from 2 to 11 and even temperature from 70 to 100°C; These advantages are facilitated with the pharmaceutical industry (Balty et al, 2020 ; Roblin et al, 2020 ).…”