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

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

Crystallographic snapshots of a B12-dependent radical SAM methyltransferase

International audienceAbstract By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas 1,2 . The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications 3–6 , such as a unique C -methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S- adenosyl- l -methionine (SAM) enzyme 7,8 . Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans , a unique B 12 (cobalamin)-dependent radical SAM enzyme 9 . The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme–substrate complex offers a glimpse into a B 12 -dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B 12 -dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry

Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase

International audienceLife relies on countless chemical reactions, almost all of which need to be sped up by enzymes. About half of all enzymes carry metal ions that expand the range of the reactions that they can catalyze. In some enzymes these metal ions assemble with sulfur ions to form so-called metalloclusters. These structures can carry out many different types of reactions, including converting simple forms of elements like nitrogen and carbon into other forms that can be used to make more complicated biological molecules.One enzyme that contains metalloclusters is carbon monoxide dehydrogenase. Known as CODH for short, this enzyme uses a metallocluster called the “C-cluster” to interconvert two gases: the pollutant carbon monoxide and the greenhouse gas carbon dioxide. CODH enzymes are found inside certain bacteria, but they are also of interest for humans, who wish to use them to remove the harmful gases from the environment. But this is not as simple as it may at first seem: CODH enzymes usually become inactive when exposed to air because the metalloclusters fall apart in the presence of oxygen. One CODH enzyme from a widespread bacterium called Desulfovibrio vulgaris, however, is an attractive target for industrial use because it can tolerate oxygen better. Yet, it is still unclear why this enzyme does not get inactivated the way other CODHs do.Wittenborn et al. have now characterized the CODH enzyme from D. vulgaris in more depth via a technique called X-ray crystallography, which can reveal the location of individual atoms within a molecule. By a happy accident, the structures revealed that the C-cluster can adopt a dramatically different arrangement of metal and sulfur ions after being exposed to oxygen. This rearrangement is fully reversible; when oxygen is removed, the metal and sulfur ions move back to their normal positions. This ability to flip between different arrangements appears to protect the metallocluster from losing its metal ions when exposed to oxygen.By providing structural snapshots of how CODH responds to oxygen these results provide a more complete understanding of an enzyme that plays a key role in the global carbon cycle. This understanding could help scientists to develop bioremediation tools to remove carbon monoxide and carbon dioxide from the atmosphere and to engineer bacteria to capture carbon to make biofuels

Interaction of the H-cluster of FeFe hydrogenase with halides

International audienceFeFe hydrogenases catalyse H2 oxidation and production using a "H-cluster", where two Fe ions are bound by an aza-dithiolate (adt) ligand. Various hypotheses have been proposed (by us and others) to explain that the enzyme reversibly inactivates under oxidizing, anaerobic conditions: intramolecular binding of the N atom of adt, formation of the so-called Hox/inact state or non-productive binding of H2 to isomers of the H-cluster. Here we show that none of the above explains the new finding that the anaerobic, oxidative, H2-dependent reversible inactivation is strictly dependent on the presence of Cl- or Br-. We provide experimental evidence that chloride uncompetitively inhibits the enzyme: it reversibly binds to catalytic intermediates of H2 oxidation (but not to the resting "Hox" state), after which oxidation locks the active site into a stable, saturated, inactive form, the structure of which is proposed here based on DFT calculations. The halides interact with the amine group of the H-cluster but do not directly bind to iron. It should be possible to stabilize the inhibited state in amounts compatible with spectroscopic investigations to explore further this unexpected reactivity of the H-cluster of hydrogenase

Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme

International audienced-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-l-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, size-exclusion chromatography–small-angle x-ray scattering, electron paramagnetic resonance spectroscopy and biochemical analyses reveal how epimerizations are installed in RiPPs and support an unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products

Photoinhibition of FeFe Hydrogenase

International audienceIn the enzyme FeFe hydrogenase, hydrogen oxidation and production occur at the H-cluster, a Fe​ 6​ S​ 6 active site that bears intrinsic carbonyl and cyanide ligands. This enzyme has been coupled to photosensitizers to design H​ 2 photoproduction systems, and yet, according to earlier reports, the enzyme from ​ Desulfovibrio desulfuricans is "easily destroyed" in "normal laboratory light". Here we report direct electrochemistry measurements of the effect of light on the activity of the enzymes from ​ Chlamydomonas reinhardtii and ​ Clostridium acetobutylicum, together with TDDFT and DFT calculations of the reactivity of the excited states of the H-cluster. We conclude that visible light does not inhibit these enzymes, but absorption of UV-B (280-315 nm) irreversibly damages the H-cluster by triggering the release of an intrinsic CO ligand; the resulting unsaturated species rearranges and protonates to form a stable, inactive dead-end. Answering the question of which particular hydrogenase can resist which particular wavelengths is important regarding solar H​ 2 production, and our results show that some but not all FeFe hydrogenases can actually be combined with photosensitizers that utilise the solar spectrum, provided a UV screen is used. We suggest that further investigations of the compatibility of hydrogenases or hydrogenase mimics with light-harvesting systems should also consider the possibility of irreversible​ ​ photoinhibition