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

Clémence Balty,Alain Guillot,Laura Fradale,Clémence Brewee,Benjamin Lefranc,Christian Herrero,Corine Sandström,Jérôme Leprince,Olivier Berteau,Alhosna Benjdia

doi:10.1074/jbc.ra120.015371

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.

Highlights

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood
To gain insights into the role of the ribosomally synthesized and posttranslationally modified peptides (RiPPs) precursor peptide recognition element (RRE) and SPASM domains and thioether bond catalysis, we undertook the biochemical characterization of RumMC2 that we have recently shown installs four Ca-thioether bridges in Ruminococcin C (RumC) [7] (Fig. 1a)
Identified in the anaerobic sulfatase-maturating enzyme which catalyzes the oxidation of a critical catalytic residue in sulfatases [37, 38, 43,44,45,46], this domain is widespread among radical SAM enzymes catalyzing peptide posttranslational modification such AlbA [32, 33], KW cyclase [10, 47], and PqqE [48]

Summary

Introduction

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. Early studies have shown that radical SAM enzymes involved in protein [37, 38] or peptide posttranslational modification often possess a so-called SPASM/ twitch domain containing auxiliary iron-sulfur clusters [39,40,41].

Results

Conclusion