Characterization of an Ntn-hydrolase, PvdQ, and an L-ornithine N5-monooxygenase, PvdA, involved in pyoverdine biosynthesis in Pseudomonas aeruginosa PAO1

Pyoverdine is a siderophore and virulence factor produced by Pseudomonas aeruginosa, an opportunistic pathogen affecting the immunocompromised. Previous attempts to disrupt pyoverdine synthesis have resulted in pyoverdine negative phenotypes and a subsequent reduction in P. aeruginosa virulence. The components of the pyoverdine biosynthesis pathway therefore represent attractive antibiotic targets, however, not all aspects of this pathway are understood. This thesis investigated two enzymes involved in pyoverdine synthesis in an attempt to increase understanding of the biosynthesis process.;We sought to increase the structure -- functional understanding of PvdA, an L-ornithine N5-hydroxylase involved in the derivation of pyoverdine peptide amino acids, via mutagenesis of genes encoding several residues conserved amongst N-hydroxylating monooxygenases. The positive charge of the K69 residue appears to be important for substrate binding as an alanine substitution exhibited unusual kinetics in that at least 6 mM of L-ornithine is required to enhance its NADPH oxidation activity. A 5-fold decrease in NADPH oxidation activity in the presence of L-ornithine, was also observed, which could be somewhat restored with an arginine variant. H189 appears to be important to catalysis as an alanine variant had a 12-fold decrease in NADPH oxidation activity compared to wild-type PvdA as well as an approximately 3000-fold decrease in the rate of formation of the hydroperoxyflavin production.;PvdQ is an Ntn-hydrolase with an unknown function that has been implicated in pyoverdine biosynthesis. Previous pvdQ knockout studies have resulted in the disruption of pyoverdine production, however, intact gene complementation was not completed. pvdQ complementation was completed in this thesis illustrating that lack of pyoverdine production was due to the pvdQ knockout mutation. Unsuccessful attempts were made to overexpress and purify PvdQ. Expression in E. coli resulted in insoluble proteins and there was no significant expression in P. aeruginosa.