flavin-adenine-dinucleotide and pyoverdin

Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism.

Topics: Animals; Chlorides; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Hydroxylation; Kinetics; Lysine; Microsomes, Liver; Mixed Function Oxygenases; NADP; Oligopeptides; Ornithine; Oxidation-Reduction; Parabens; Pseudomonas aeruginosa; Pseudomonas fluorescens; Schizosaccharomyces; Siderophores; Solubility; Substrate Specificity; Swine

Pseudomonas aeruginosa is an opportunistic pathogen that produces the siderophore pyoverdine, which enables it to acquire the essential nutrient iron from its host. Formation of the iron-chelating hydroxamate functional group in pyoverdine requires the enzyme PvdA, a flavin-dependent monooxygenase that catalyzes the N(5) hydroxylation of l-ornithine. pvdA from P. aeruginosa was successfully overexpressed in Escherichia coli, and the enzyme was purified for the first time. The enzyme possessed its maximum activity at pH 8.0. In the absence of l-ornithine, PvdA has an NADPH oxidase activity of 0.24 +/- 0.02 micromol min(-1) mg(-1). The substrate l-ornithine stimulated this activity by a factor of 5, and the reaction was tightly coupled to the formation of hydroxylamine. The enzyme is specific for NADPH and flavin adenine dinucleotide (FAD(+)) as cofactors, as it cannot utilize NADH and flavin mononucleotide. By fluorescence titration, the dissociation constants for NADPH and FAD(+) were determined to be 105.6 +/- 6.0 microM and 9.9 +/- 0.3 microM, respectively. Steady-state kinetic analysis showed that the l-ornithine-dependent NADPH oxidation obeyed Michaelis-Menten kinetics with apparent K(m) and V(max) values of 0.58 mM and 1.34 micromol min(-1) mg(-1). l-Lysine was a nonsubstrate effector that stimulated NADPH oxidation, but uncoupling occurred and hydrogen peroxide instead of hydroxylated l-lysine was produced. l-2,4-Diaminobutyrate, l-homoserine, and 5-aminopentanoic acid were not substrates or effectors, but they were competitive inhibitors of the l-ornithine-dependent NADPH oxidation reaction, with K(ic)s of 3 to 8 mM. The results indicate that the chemical nature of effectors is important for simulation of the NADPH oxidation rate in PvdA.

Topics: Amino Acids, Neutral; Aminobutyrates; Cloning, Molecular; Enzyme Inhibitors; Enzyme Stability; Escherichia coli; Flavin-Adenine Dinucleotide; Homoserine; Hydrogen-Ion Concentration; Hydroxylamine; Lysine; Mixed Function Oxygenases; NADP; Oligopeptides; Ornithine; Oxidation-Reduction; Pseudomonas aeruginosa; Recombinant Proteins; Siderophores; Substrate Specificity