Biosynthesis of Pseudomonas aeruginosa Natural Products

Pseudomonas aeruginosa is a pathogenic bacterium that infects many hospitalized patients with compromised immune systems. The small molecules synthesized by this bacterium contribute in part to its pathogenicity. Though many small molecules made by this bacterium are known, the genome of P. aeruginosa contains many uncharacterized gene clusters, which indicate many other small molecules have not been identified. Here I describe our efforts to identify new small molecules made by P. aeruginosa and characterize the enzymatic reactions that synthesize these small molecules. The biosynthesis of the antimetabolite L-2-amino-4-methoxy-trans-3-butenoic-acid (AMB) is described. I show that AMB is first constructed as an alanyl-AMB dipeptide. The order and timing of modifications on the nonribosomal peptide synthetase (NRPS)-bound substrates is identified using a chemical capture technique. Analysis of the intermediates of AMB biosynthesis reveal a cryptic α,β-dehydroamino acid intermediate. I identified the enzyme responsible for this dehydration as an unusual condensation (C) domain in the NRPS AmbE. A crystal structure was obtained of this domain and key residues for the function of this dehydrating C domain were identified. Using the dehydrating C domain as a biosynthetic hook to discover new natural products, a dehydrating C domain is shown to be involved in the synthesis of another P. aeruginosa natural product azabicyclene as well as many other unknown natural products in a variety of bacteria. Azabicyclene is synthesized under quorum sensing conditions and has no known function. The biosyntheses of two other natural product antibiotics are described: bicyclomycin and fluopsin C. Both antibiotics were known to be produced by other bacteria but not in P. aeruginosa. I demonstrate the fluopsin C is synthesized via the forging of two cysteines into minimal ligands which bind a copper ion, and bicyclomycin is made via successive oxidations of an isoleucine-leucine cyclodipeptide. Studying the small molecule inventory of P. aeruginosa will reveal the strategies that this pathogen uses to survive in many environments, such as immunocompromised individuals.Doctor of Philosoph