Stéphane Grimaldi

Methyl-coenzyme M reductase (MCR) is a crucial enzyme for methanogenesis and harbors several unusual post-translational modifications. Recent studies have identified glutamine C-methyltransferase (QCMT), as a B₁₂-dependent radical SAM enzyme responsible for methylating a glutamine residue within the MCR active site. B₁₂-dependent radical SAM enzymes have the remarkable ability to alkylate unactivated Csp²- and Csp³-atoms in a stereoselective manner. However, the factors influencing the stereo-selectivity and catalytic properties of this emerging superfamily of enzymes remain poorly understood. Here, we report the mechanistic, structural, and biochemical investigation of several QCMTs. Our findings reveal significant differences among them, notably in their ability to bind cobalamin. In addition, our data support that Cα H-atom abstraction and methyl transfer are not concerted but rather independent processes that require motion within the enzyme's active site. We also demonstrate that QCMT catalyzes novel reactions, including the formation of unnatural C-methylated residues, peptide epimerization, reversible H-atom abstraction, and the direct conversion of glycine into D-alanine. Overall, our data are consistent with QCMT being a unique and versatile biocatalyst allowing for the installation of unnatural post-translational modifications and provide a structural and biochemical rationale for the control of the stereochemistry by B₁₂-dependent radical SAM enzymes.

Methyl-coenzyme M reductase (MCR) is a crucial enzyme for methanogenesis and harbors several unusual post-translational modifications. Recent studies have identified glutamine C-methyltransferase (QCMT), as a B ₁₂ -dependent radical SAM enzyme responsible for methylating a glutamine residue within the MCR active site. B ₁₂ -dependent radical SAM enzymes have the remarkable ability to alkylate unactivated C sp ² - and Csp ³ -atoms in a stereoselective manner. However, the factors influencing the stereo-selectivity and catalytic properties of this emerging superfamily of enzymes remain poorly understood. In this study, we report the mechanistic, structural, and biochemical investigation of several QCMTs. Our findings reveal significant differences among them, notably in their ability to bind cobalamin. In addition, our data support that Cα H-atom abstraction and methyl transfer are not concerted but rather independent processes that require motion within the enzyme’s active site. We also demonstrate that QCMT can catalyze novel reactions, including the formation of unnatural C -methylated residues, peptide epimerization, reversible H-atom abstraction, and the direct conversion of glycine into d -alanine. Overall, our data are consistent with QCMT being a unique and versatile biocatalyst allowing for the installation of unnatural post-translational modifications and provide a structural and biochemical rationale for the control of the stereochemistry by B ₁₂ -dependent radical SAM enzymes.

Radical SAM enzymes are the most widespread biocatalysts. These metalloenzymes, using S-adenosyl-l-methionine (SAM) and a [4Fe-4S] cluster as central cofactors, catalyze a broad range of chemically challenging transformations. The vast majority of radical SAM enzymes initiate their reaction by the homolytic cleavage of the SAM C5'-S bond and the generation of the central 5'-deoxyadenosyl radical (5'-dA·). In this study, by combining spectroscopic approaches with labeling and biochemical analyses, we show that ArsL, the key enzyme in the biosynthesis of the arsenic-containing antibiotic arsinothricin, catalyzes a unique reaction: the addition of the 3-amino-3-carboxypropyl radical (ACP·) to As(III). Remarkably, by exploiting several radical trapping strategies, we demonstrate that in sharp contrast to canonical radical SAM enzymes ArsL cleaves the SAM Cγ-S bond. In addition, using electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopies, we establish that ArsL has a unique SAM binding mode, consistent with its catalytic properties and predicted structure. Notably, EPR and HYSCORE analyses support that SAM interacts with the radical SAM [4Fe-4S] cluster in an uncharacteristic conformation to form ACP·. Collectively, our study reveals that members of the superfamily of radical SAM enzymes are able to finely tune the binding of the SAM cofactor in order to perform unique chemistries.

Mono-pyranopterin-containing sulfite-oxidizing enzymes (SOEs), including eukaryotic sulfite oxidases and homologous prokaryotic sulfite dehydrogenases (SDHs), are molybdenum enzymes that exist in almost all forms of life, where they catalyze the direct oxidation of sulfite into sulfate, playing a key role in protecting cells and organisms against sulfite-induced damage. To decipher their catalytic mechanism, we have previously provided structural and spectroscopic evidence for direct coordination of HPO4 2- to the Mo atom at the active site of the SDH from the hyperthermophilic bacterium Thermus thermophilus (TtSDH), mimicking the proposed sulfate-bound intermediate proposed to be formed during catalysis. In this work, by solving the X-ray crystallographic structure of the unbound enzyme, we resolve the changes in the hydrogen bonding network in the molybdenum environment that enable the stabilization of the previously characterized phosphate adduct. In addition, electron paramagnetic resonance spectroscopic study of the enzyme over a wide pH range reveals the formation of pH-dependent Mo(V) species, a characteristic feature of eukaryotic SOEs. The combined use of HYSCORE, H2O/D2O exchange, and density functional theory calculations allows the detailed characterization of a typical low pH Mo(V) species previously unreported in bacterial SOEs, underlining the conservation of the active site properties of SOEs irrespective of their source organism.

D-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.

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 B₁₂ (cobalamin)-dependent radical SAM enzyme⁹. 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 B₁₂-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 B₁₂-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.