biostudies-literatureMorgan GLEunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNICHD NIH HHSNCI NIH HHSUniversity of North Carolina at Chapel HillRita Allen FoundationDavid and Lucile Packard FoundationNIGMS NIH HHS2776-2786https://www.ebi.ac.uk/biostudies/studies/S-EPMC8917869biostudies-literatureUnknown16(12)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 <i>Pseudomonas virulence factor</i> (<i>pvf</i>) 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 <i>N</i>-oxides that require an additional <i>N-</i>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 <i>pvf</i>-encoded enzymes sets the stage for further understanding the functions of <i>pvf</i> in bacterial cell-to-cell signaling.ACS chemical biologyEnzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase.PMC8917869T32 GM008570P30 CA016086DP2 HD094657DP2HD094657Aube JCrawford DMMorgan GLLi BLi KfalseEnzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase.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 <i>Pseudomonas virulence factor</i> (<i>pvf</i>) 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 <i>N</i>-oxides that require an additional <i>N-</i>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 <i>pvf</i>-encoded enzymes sets the stage for further understanding the functions of <i>pvf</i> in bacterial cell-to-cell signaling.2021-01-01T00:00:00Z2021 Dec2025-04-26T18:45:22.837Z2025-04-06T15:52:03.71ZS-EPMC89178693476771210.1021/acschembio.1c00623