Overexpression of the Pseudomonas virulence factor ( pvf) biosynthetic operon led to the identification of a family of pyrazine N-oxides (PNOs), including a novel dihydropyrazine N,N'-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)PNOs was characterized, and a biosynthetic pathway for (d)PNOs was proposed. This work highlights the unique chemistry catalyzed by pvf-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway.

The Pseudomonas virulence factor (pvf) biosynthetic operon has been implicated in bacterial virulence and signaling. We identified 308 bacterial strains containing pvf homologues that likely produce signaling molecules with distinct structures and biological activities. Several homologues of the nonribosomal peptide synthetase (NRPS), PvfC, were biochemically characterized and shown to activate L-Val or L-Leu. The amino acid selectivity of PvfC and its homologues likely direct pvf signaling activity. We explored the natural diversity of the active site residues present in 92% of the adenylation domains of PvfC homologues and identified key residues for substrate selection and catalysis. Sequence similarity network (SSN) analysis revealed grouping of PvfC homologues that harbor the same active site residues and activate the same amino acids. Our work identified PvfC as a gatekeeper for the structure and bioactivity of the pvf-produced signaling molecules. The combination of active site residue identification and SSN analysis can improve the prediction of aliphatic amino acid substrates for NRPS adenylation domains. Communication pubs.acs.org/biochemistry

The Pseudomonas virulence factor (pvf) operon is essential for the biosynthesis of two very different natural product scaffolds: the (dihydro)pyrazine-N-oxides and the diazeniumdiolate, valdiazen. PvfB is a member of the nonheme diiron N-oxygenase enzyme family that commonly convert anilines to their nitroaromatic counterparts. In contrast, we show that PvfB catalyzes N-oxygenation of the aamine of valine, first to the hydroxylamine and then the nitroso, while linked to the carrier protein of PvfC. PvfB modification of PvfC-tethered valine was observed directly by protein NMR spectroscopy, establishing the intermediacy of the hydroxylamine. This work reveals a central role for PvfB in the biosynthesis of (dihydro)pyrazine-N-oxides and valdiazen. ThePseudomonas virulence factor (pvf, Figure 1 A), a widely conserved operon in proteobacteria, [1] plays important roles in virulence and bacterial cell-to-cell signaling. [2] This cluster, first reported from Pseudomonas entomophila L48, has now been identified in more than 500 strains (Supporting Information, Dataset S1). [1] We showed that pvf-encoded enzymes from P. entomophila L48 produce a novel family of molecules-(dihydro)pyrazine-N-oxides [(d)PNOs, Figure 1 B)]. Concurrently, the ham operon, a pvf-like cluster from Burkholderia cenocepacia H111, was implicated in the production of a very different molecule, valdiazen-a valinol diazeniumdiolate (Figure 1 B). [3] The (d)PNOs and valdiazen both contain unusual NO functionalities but differ in overall structure. We set out to understand the biosynthetic chemistries for these different compounds. Pvf commonly comprises four genes: a nonribosomal peptide synthetase (NRPS, pvfC or hamD), a non-heme diiron enzyme (pvfB or hamC), and two genes of unknown function, pvfA/hamA and pvfD/hamE (Figure 1 A). The ham operon contains a fifth gene, hamB, encoding a putative cupin protein. Both pvfC and pvfB from P. entomophila are necessary to produce the (d)PNOs. [4] NRPSs selectively activate and tether amino or carboxylic acids to thiolation domains where substrates undergo modifications by tailoring enzymes. [5] The NPRS PvfC activates and tethers l-valine to its thiolation domain (Figure 1 B), [1, 4] and similar chemistry could be predicted for HamD. The role of the putative diiron N-oxygenases PvfB or HamC, however, remained unclear.

Cell-to-cell communication via chemical signals is an essential mechanism that pathogenic bacteria use to coordinate group behaviors and promote virulence. The Pseudomonas virulence factor (pvf) gene cluster is distributed in more than 500 strains of proteobacteria including both plant and human pathogens. The pvf cluster has been implicated in the production of signaling molecules important for virulence; however, the regulatory impact of these signaling molecules on virulence had not been elucidated. Using the insect pathogen Pseudomonas entomophila L48 as a model, we demonstrated that pvf-encoded biosynthetic enzymes produce PVF autoinducers that regulate the expression of pvf genes and a gene encoding the toxin monalysin via quorum sensing. In addition, PVF autoinducers regulate the expression of nearly 200 secreted and membrane proteins, including toxins, motility proteins, and components of the type VI secretion system, which play key roles in bacterial virulence, colonization, and competition with other microbes. Deletion of pvf also altered the secondary metabolome. Six major compounds upregulated by PVF autoinducers were isolated and structurally characterized, including three insecticidal 3-indolyl oxazoles, the labradorins, and three antimicrobial pyrrolizidine alkaloids, the pyreudiones. The signaling properties of PVF autoinducers and their wide-ranging regulatory effects indicate multifaceted roles of PVF in controlling cell physiology and promoting virulence. The broad genome distribution of pvf suggests that PVF-mediated signaling is relevant to many bacteria of agricultural and biomedical significance.

Nonribosomal peptide synthetases (NRPSs) are typically multimodular enzymes that assemble amino acids or carboxylic acids into complex natural products. Here, we characterize a monomodular NRPS, PvfC, encoded by the Pseudomonas virulence factor (pvf) gene cluster that is essential for virulence and signaling in different bacterial species. PvfC exhibits a unique adenylation-thiolation-reductase (ATR) domain architecture that is understudied in bacteria. We show that the activity of PvfC is essential in the production of seven leucine-derived heterocyclic natural products, including two pyrazines, a pyrazinone, and a rare disubstituted imidazole, as well as three pyrazine N-oxides that require an additional N-oxygenation step. Mechanistic studies reveal that PvfC, without a canonical peptide-forming domain, makes a dipeptide aldehyde intermediate en route to both the pyrazinone and imidazole. Our work identifies a novel biosynthetic route for the production of pyrazinones, an emerging class of signaling molecules and virulence factors. Our discovery also showcases the ability of monomodular NRPSs to generate amino acid- and dipeptide-aldehydes that lead to diverse natural products. The diversity-prone biosynthesis by the pvf-encoded enzymes sets the stage for further understanding the functions of pvf in bacterial cell-to-cell signaling.