Elucidating the Biosyntheses of Oxyvinylglycine and Pyrrolizidine Alkaloid Natural Products from Pseudomonas

Bacteria offer an incredibly diverse variety of bioactive secondary metabolites, or natural products. Studying natural products involves identifying compounds, linking their production to biosynthetic gene clusters, and characterizing their biosynthetic pathways. Biosynthetic enzymes often catalyze unique chemical modifications that are of interest to both academia and industry. Pseudomonas fluorescens is a plant-associated bacterium that produces several natural products that possess crop-protective or growth-modifying activity. Pseudomonas aeruginosa is a human pathogen that is heavily studied due to its drug resistance, widespread presence in clinical settings, and propensity to cause serious illness in immunocompromised people. In this dissertation, I discuss my efforts to characterize the biosynthetic pathways and enzymes involved in the biosyntheses of natural products from both of these pseudomonads. Three compounds produced by P. fluorescens WH6 are nonproteinogenic amino acids classified as oxyvinylglycines (OVGs). They are encoded by the gvg gene cluster and are unique to the OVG family, as they contain a rare vinyl O-N bond in their side chain. I show that the gvg products are derived from a homoserine precursor and that the product AOVG is likely not a branching point in the biosynthesis of the three compounds. I also characterize the activity of two Gvg enzymes, demonstrating that the iron-dependent enzyme GvgC is the origin of the N-O bonds and that GvgI is an amino acid N-formyltransferase. A highly conserved gene cluster in P. aeruginosa PAO1 called aze has homology to other bacterial clusters that produce pyrrolizidine alkaloid (PA) natural products. The gene clusters encode unique nonribosomal peptide synthetases that dehydrate and cyclize dipeptides, and Baeyer-Villiger monooxygenases that catalyze ring expansions and contractions of their substrates. I characterize the activity of three Aze enzymes in vitro and show that the oxidoreductase AzeE modifies an early-stage intermediate of the monooxygenase AzeC. I also show that AzeB, AzeC, and AzeE accept both proline-derived and azetidine-2-carboxylic acid-derived substrates.These enzymes produce intermediates en route to a newly discovered natural product scaffold that selectively inhibits ClpP proteases.