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The combination of next-generation DNA sequencing technologies and bioinformatics have revitalized natural product discovery. Using a new bioinformatic search strategy, we recently identified ~600 gene clusters in animal microbiomes that code for ribosomal peptide natural products synthesized by radical S-adenosylmethionine enzymes. These grouped into 16 subfamilies and pointed to an unexplored microbiome biosynthetic landscape. Herein, we report the structure, biosynthesis, and function of one of these natural product groups, that we term enteropeptins, from the gut microbe Enterococcus cecorum. We elucidate three novel reactions, each catalyzed by a different family of metalloenzymes, in the biosynthesis of enteropeptins. Among these, we characterize the founding member of a widespread superfamily of Fe-S-containing methyltransferases, which, together with a di-Mn-dependent arginase, installs an N-methylornithine in the peptide sequence. Biological assays with the mature product revealed bacteriostatic activity only against the producing strain, extending an emerging theme of fratricidal or self-inhibitory metabolites in microbiome firmicutes.

Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic β-ether and a heterocyclic sp

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products that harbor diverse chemical functionalities, usually introduced via the action of a small number of tailoring enzymes. We have been interested in RiPP biosynthetic gene clusters that encode unusual metalloenzymes, as these may install as yet unknown alterations. Using a new bioinformatic search strategy, we recently identified an array of unexplored RiPP gene clusters that are quorum sensing-regulated and contain one or more uncharacterized radical S-adenosylmethionine (RaS) metalloenzymes. Herein, we investigate the reaction of one of these RaS enzymes and find that it installs an intramolecular β-thioether bond onto its substrate peptide by connecting a Cys-thiol group to the β-carbon of an upstream Asn residue. The enzyme responsible, NxxcB, accepts several amino acids in place of Asn and introduces unnatural β-thioether linkages at unactivated positions. This new transformation adds to the growing list of Nature's peptide macrocyclization strategies and expands the already impressive catalytic repertoire of the RaS enzyme superfamily.

Peptide natural products are often used as signals or antibiotics and contain unusual structural modifications, thus providing opportunities for expanding our understanding of Nature's therapeutic and biosynthetic repertoires. Herein, we have investigated the under-explored biosynthetic potential of Streptococci, prevalent bacteria in mammalian microbiomes that include mutualistic, commensal, and pathogenic members. Using a new bioinformatic search strategy, in which we linked the versatile radical S-adenosylmethionine (RaS) enzyme superfamily to an emerging class of natural products in the context of quorum sensing control, we identified numerous, uncharted biosynthetic loci. Focusing on one such locus, we identified an unprecedented post-translational modification, consisting of a tetrahydro[5,6]benzindole cyclization motif in which four unactivated positions are linked by two C-C bonds in a regio- and stereospecific manner by a single RaS enzyme. Our results expand the scope of reactions that microbes have at their disposal in concocting complex ribosomal peptides.

The AraC/XylS-family transcriptional regulator ToxT is the master virulence activator of Vibrio cholerae, the gram-negative bacterial pathogen that causes the diarrheal disease cholera. Unsaturated fatty acids (UFAs) found in bile inhibit the activity of ToxT. Crystal structures of inhibited ToxT bound to UFA or synthetic inhibitors have been reported, but no structure of ToxT in an active conformation had been determined. Here we present the 2.5 Å structure of ToxT without an inhibitor. The structure suggests release of UFA or inhibitor leads to an increase in flexibility, allowing ToxT to adopt an active conformation that is able to dimerize and bind DNA. Small-angle X-ray scattering was used to validate a structural model of an open ToxT dimer bound to the cholera toxin promoter. The results presented here provide a detailed structural mechanism for virulence gene regulation in V. cholerae by the UFA components of bile and other synthetic ToxT inhibitors.

The biosynthetic pathways of microbial natural products provide a rich source of novel enzyme-catalyzed transformations. Using a new bioinformatic search strategy, we recently identified an abundance of gene clusters for ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain at least one radical S-adenosylmethionine (RaS) metalloenzyme and are regulated by quorum sensing. In the present study, we characterize a RaS enzyme from one such RiPP gene cluster and find that it installs an aliphatic ether cross-link at an unactivated carbon center, linking the oxygen of a Thr side chain to the α-carbon of a Gln residue. This reaction marks the first ether cross-link installed by a RaS enzyme. Additionally, it leads to a new heterocyclization motif and underlines the utility of our bioinformatics approach in finding new families of RiPP modifications.

Environmental exposure is a key factor of understanding health and diseases. Beyond genetic propensities, many disorders are, in part, caused by human interaction with harmful substances in the water, the soil, or the air. Limited data is available on a disease or substance basis. However, we compile a global repository from literature surveys matching environmental chemical substances exposure with human disorders. We build a bipartite network linking 60 substances to over 150 disease phenotypes. We quantitatively and qualitatively analyze the network and its projections as simple networks. We identify mercury, lead and cadmium as associated with the largest number of disorders. Symmetrically, we show that breast cancer, harm to the fetus and non-Hodgkin's lymphoma are associated with the most environmental chemicals. We conduct statistical analysis of how vertices with similar characteristics form the network interactions. This dyadicity and heterophilicity measures the tendencies of vertices with similar properties to either connect to one-another. We study the dyadic distribution of the substance classes in the networks show that, for instance, tobacco smoke compounds, parabens and heavy metals tend to be connected, which hint at common disease causing factors, whereas fungicides and phytoestrogens do not. We build an exposure network at the systems level. The information gathered in this study is meant to be complementary to the genome and help us understand complex diseases, their commonalities, their causes, and how to prevent and treat them.