Bacterial Autoimmune Drug Metabolism Transforms an Immunomodulator into Structurally and Functionally Divergent Antibiotics.

Tapinarof is a stilbene drug that is used to treat psoriasis and atopic dermatitis, and is thought to function through regulation of the AhR and Nrf2 signaling pathways, which have also been linked to inflammatory bowel diseases. It is produced by the gammaproteobacterial Photorhabdus genus, which thus represents a model to probe tapinarof structural and functional transformations. We show that Photorhabdus transforms tapinarof into novel drug metabolism products that kill inflammatory bacteria, and that a cupin enzyme contributes to the conversion of tapinarof and related dietary stilbenes into novel dimers. One dimer has activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), and another undergoes spontaneous cyclizations to a cyclopropane-bridge-containing hexacyclic framework that exhibits activity against Mycobacterium. These dimers lack efficacy in a colitis mouse model, whereas the monomer reduces disease symptoms.

Characterization of a Hybrid Nonribosomal Peptide-Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens.

Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-ß-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-ß-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

Functional Characterization of a Condensation Domain That Links Nonribosomal Peptide and Pteridine Biosynthetic Machineries in Photorhabdus luminescens.

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of biologically important small molecules. NRPSs can interface with other enzymes to form hybrid biosynthetic systems that expand the structural and functional diversity of their products. The pepteridines are metabolites encoded by an unprecedented pteridine-NRPS-type hybrid biosynthetic gene cluster in Photorhabdus luminescens, but how the distinct enzymatic systems interface to produce these molecules has not been examined at the biochemical level. By an unknown mechanism, the genetic locus can also affect the regulation of other enzymes involved in autoinducer and secondary metabolite biosynthesis. Here, through in vitro protein biochemical assays, we demonstrate that an atypical NRPS condensation (C) domain present in the pathway condenses acyl units derived from α-keto acids onto a free 5,6,7,8-tetrahydropterin core, producing the tertiary cis-amide-containing pepteridines. Solution studies of the chemically synthesized molecules led to the same amide regiochemistries that were observed in the natural products. The biochemical transformations mediated by the C domain destroy the radical scavenging activity of its redox active tetrahydropterin substrate. Secondary metabolite analyses revealed that the pepteridine locus affects select metabolic pathways associated with quorum sensing, antibiosis, and symbiosis. Taken together, the results suggest that the pathway likely regulates cellular redox and specialized metabolic pathways through engagement with the citric acid cycle.

Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster.

Nonribosomal peptides represent a large class of metabolites with pharmaceutical relevance. Pteridines, such as pterins, folates, and flavins, are heterocyclic metabolites that often serve as redox-active cofactors. The biosynthetic machineries for construction of these distinct classes of small molecules operate independently in the cell. Here, we discovered an unprecedented nonribosomal peptide synthetase-like-pteridine synthase hybrid biosynthetic gene cluster in Photorhabdus luminescens using genome synteny analysis. P. luminescens is a Gammaproteobacterium that undergoes phenotypic variation and can have both pathogenic and mutualistic roles. Through extensive gene deletion, pathway-targeted molecular networking, quantitative proteomic analysis, and NMR, we show that the genetic locus affects the regulation of quorum sensing and secondary metabolic enzymes and encodes new pteridine metabolites functionalized with cis-amide acyl-side chains, termed pepteridine A (1) and B (2). The pepteridines are produced in the pathogenic phenotypic variant and represent the first reported metabolites to be synthesized by a hybrid NRPS-pteridine pathway. These studies expand our view of the combinatorial biosynthetic potential available in bacteria.

Stilbene epoxidation and detoxification in a Photorhabdus luminescens-nematode symbiosis.

Members of the gammaproteobacterial Photorhabdus genus share mutualistic relationships with Heterorhabditis nematodes, and the pairs infect a wide swath of insect larvae. Photorhabdus species produce a family of stilbenes, with two major components being 3,5-dihydroxy-4-isopropyl-trans-stilbene (compound 1) and its stilbene epoxide (compound 2). This family of molecules harbors antimicrobial and immunosuppressive activities, and its pathway is responsible for producing a nematode "food signal" involved in nematode development. However, stilbene epoxidation biosynthesis and its biological roles remain unknown. Here, we identified an orphan protein (Plu2236) from Photorhabdus luminescens that catalyzes stilbene epoxidation. Structural, mutational, and biochemical analyses confirmed the enzyme adopts a fold common to FAD-dependent monooxygenases, contains a tightly bound FAD prosthetic group, and is required for the stereoselective epoxidation of compounds 1 and 2. The epoxidase gene was dispensable in a nematode-infective juvenile recovery assay, indicating the oxidized compound is not required for the food signal. The epoxide exhibited reduced cytotoxicity toward its producer, suggesting this may be a natural route for intracellular detoxification. In an insect infection model, we also observed two stilbene-derived metabolites that were dependent on the epoxidase. NMR, computational, and chemical degradation studies established their structures as new stilbene-l-proline conjugates, prolbenes A (compound 3) and B (compound 4). The prolbenes lacked immunosuppressive and antimicrobial activities compared with their stilbene substrates, suggesting a metabolite attenuation mechanism in the animal model. Collectively, our studies provide a structural view for stereoselective stilbene epoxidation and functionalization in an invertebrate animal infection model and provide new insights into stilbene cellular detoxification.

Activating and Attenuating the Amicoumacin Antibiotics.

The amicoumacins belong to a class of dihydroisocoumarin natural products and display antibacterial, antifungal, anticancer, and anti-inflammatory activities. Amicoumacins are the pro-drug activation products of a bacterial nonribosomal peptide-polyketide hybrid biosynthetic pathway and have been isolated from Gram-positive Bacillus and Nocardia species. Here, we report the stimulation of a "cryptic" amicoumacin pathway in the entomopathogenic Gram-negative bacterium Xenorhabdus bovienii, a strain not previously known to produce amicoumacins. X. bovienii participates in a multi-lateral symbiosis where it is pathogenic to insects and mutualistic to its Steinernema nematode host. Waxmoth larvae are common prey of the X. bovienii-Steinernema pair. Employing a medium designed to mimic the amino acid content of the waxmoth circulatory fluid led to the detection and characterization of amicoumacins in X. bovienii. The chemical structures of the amicoumacins were supported by 2D-NMR, HR-ESI-QTOF-MS, tandem MS, and polarimeter spectral data. A comparative gene cluster analysis of the identified X. bovienii amicoumacin pathway to that of the Bacillus subtilis amicoumacin pathway and the structurally-related Xenorhabdus nematophila xenocoumacin pathway is presented. The X. bovienii pathway encodes an acetyltransferase not found in the other reported pathways, which leads to a series of N-acetyl-amicoumacins that lack antibacterial activity. N-acetylation of amicoumacin was validated through in vitro protein biochemical studies, and the impact of N-acylation on amicoumacin's mode of action was examined through ribosomal structural analyses.

In vitro and in vivo single-molecule fluorescence imaging of ribosome-catalyzed protein synthesis.

Combined with the availability of highly purified, fluorescently labeled in vitro translation systems, the advent of single-molecule fluorescence imaging has ushered in a new era in high-resolution mechanistic studies of ribosome-catalyzed protein synthesis, or translation. Together with ensemble biochemical investigations of translation and structural studies of functional ribosomal complexes, in vitro single-molecule fluorescence imaging of protein synthesis is providing unique mechanistic insight into this fundamental biological process. More recently, rapidly evolving breakthroughs in fluorescence-based molecular imaging in live cells with sub-diffraction-limit spatial resolution and ever-increasing temporal resolution provide great promise for conducting mechanistic studies of translation and its regulation in living cells. Here we review the remarkable recent progress that has been made in these fields, highlight important mechanistic insights that have been gleaned from these studies thus far, and discuss what we envision lies ahead as these approaches continue to evolve and expand to address increasingly complex mechanistic and regulatory aspects of translation.