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The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus. This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified pep-tide (RiPP) with potent activity against the human pathogen Clostridium perfringens. By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to-␣-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features , and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus. This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens. By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to–α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryo-electron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

International audience; Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of bioactive peptides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of post-translational modifications catalyzed by novel radical S-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a C-to-N directional manner. By combining mutagenesis experiments with labeling studies and investigating the enzyme substrate promiscuity, we deciphered in detail the mechanism of PoyD. We notably identified a critical cysteine residue as a likely key H atom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addition, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with unnatural epimerization patterns.

A series of Gs protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β2-Adrenergic receptor (β2AR) in complex with the Gs protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular Gs protein binding site of the β2AR. Peptidomimetics were designed to mimic the 15 residue C-Terminal α-helix of the Gs protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled peptidomimetics were analysed by circular dichroism (CD) and characterised in a membrane-based cAMP accumulation assay and in a bimane fluorescence assay on purified β2AR. Several peptidomimetics inhibited agonist isoproterenol (ISO) induced cAMP formation by lowering the ISO maximal efficacy up to 61%. Moreover, some peptidomimetics were found to significantly decrease the potency of ISO up to 39-fold. In the bimane fluorescence assay none of the tested peptidomimetics could stabilise an active-like conformation of β2AR. Overall, the obtained pharmacological data suggest that some of the peptidomimetics may be able to compete with the native Gs protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation.

International audience; Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms.Background: Catalytic activity of prokaryotic and eukaryotic arylamine N-acetyltransferases (NATs) relies on a strictly conserved catalytic Cys-His-Asp triad.Results: Structural and biochemical studies identified a functional NAT with a Cys-His-Glu catalytic triad.Conclusion: The catalytic triad of these acetyltransferases is more plastic than previously believed.Significance: (BACCR)NAT3 represents the first known functional acetyltransferase with a non-canonical Cys-His-Glu catalytic triad.

Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.

ArylamineN-acetyltransferases (NATs) are xenobiotic metabolizing enzymes that catalyze the acetyl-CoA-dependent acetylation of arylamines. To better understand the mode of binding of the cofactor by this family of enzymes, the structure ofMesorhizobium lotiNAT1 [(RHILO)NAT1] was determined in complex with CoA. The F42W mutant of (RHILO)NAT1 was used as it is well expressed inEscherichia coliand displays enzymatic properties similar to those of the wild type. The apo and holo structures of (RHILO)NAT1 F42W were solved at 1.8 and 2 Å resolution, respectively. As observed in theMycobacterium marinumNAT1–CoA complex, in (RHILO)NAT1 CoA binding induces slight structural rearrangements that are mostly confined to certain residues of its `P-loop'. Importantly, it was found that the mode of binding of CoA is highly similar to that ofM. marinumNAT1 but different from the modes reported forBacillus anthracisNAT1 andHomo sapiensNAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover, it supports the notion that the presence of the `mammalian/eukaryotic insertion loop' in certain NAT enzymes impacts the mode of binding CoA by imposing structural constraints.

Mycobacterium abscessusis the most pathogenic rapid-growing mycobacterium and is one of the most resistant organisms to chemotherapeutic agents. However, structural and functional studies ofM. abscessusproteins that could modify/inactivate antibiotics remain nonexistent. Here, the structural and functional characterization of an arylamineN-acetyltransferase (NAT) fromM. abscessus[(MYCAB)NAT1] are reported. This novel prokaryotic NAT displays significantN-acetyltransferase activity towards aromatic substrates, including antibiotics such as isoniazid andp-aminosalicylate. The enzyme is endogenously expressed and functional in both the rough and smoothM. abscessusmorphotypes. The crystal structure of (MYCAB)NAT1 at 1.8 Å resolution reveals that it is more closely related toNocardia farcinicaNAT than to mycobacterial isoforms. In particular, structural and physicochemical differences from other mycobacterial NATs were found in the active site. Peculiarities of (MYCAB)NAT1 were further supported by kinetic and docking studies showing that the enzyme was poorly inhibited by the piperidinol inhibitor of mycobacterial NATs. This study describes the first structure of an antibiotic-modifying enzyme fromM. abscessusand provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity and extensive chemotherapeutic resistance ofM. abscessus.

Arylamine N-acetyltransferase (NAT) plays an important role in metabolism and detoxification of many compounds including drugs and environmental carcinogens through chemical modification of the amine group with an acetyl group. Recent studies have suggested that NATs are also involved in cancer cell growth and inhibition of the enzymes may be a potential target for cancer chemotherapy. Three-dimensional (3D) structures are available for NATs from both prokaryotes and eukaryotes. These structures provide valuable insights into the acetylation mechanism, features of the active site and the structural determinants that govern substrate/inhibitor-binding specificity. Such insights allow a more precise understanding of the structure–activity relationships for NAT substrates and inhibitors. Furthermore, the structural elucidation of NATs has generated powerful tools in the design of small molecule inhibitors that should alleviate cancer, based on the important role of the enzyme in cancer biology.

Arylamine N-acetyltransferases (NATs) are polymorphic xenobiotic metabolizing enzymes catalyzing the acetylation of aromatic amine chemicals of pharmacological/toxicological relevance (drugs, carcinogens). NATs are primordial determinants of the detoxification and/or bioactivation of these compounds. These enzymes are found in prokaryotes and eukaryotes. Several NAT isoenzymes may be present in one organism, and their substrate specificity profile and pattern of tissue expression suggest distinct functional roles.Many advances in NAT mechanism, substrate specificity, and functional impact of polymorphism have come from crystallographic and NMR studies. To date, the crystal structures of 10 different NAT homologues have been solved, including two human isoforms and several bacterial NATs. The authors present the most recent snapshot in NAT structure differences and similarities. The authors also depict the structural bases of substrate/inhibitor recognition and specificity, cofactor binding, catalytic mechanism, genetic regulation (polymorphism), and enzyme inhibition.The determination of other NATs structures will help to develop specific inhibitors of NAT enzymes with potential clinical relevance. In addition, it will contribute to the identification of endogenous substrates and novel functions associated to this family of enzymes.

This letter is a comment on Zhou et al. (2013). Arylamine N-acetyltransferases: a structural perspective. Br J Pharmacol 169: 748–760. To view this article visit http://dx.doi.org/10.1111/bph.12182