Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism.
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.
Meneely
Kathleen M
KM
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Lamb
Audrey L
AL
D000818:Animals; D002712:Chlorides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009842:Oligopeptides / Q000096:biosynthesis; D009952:Ornithine / Q000378:metabolism*; D010084:Oxidation-Reduction; D010226:Parabens / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology; D012568:Schizosaccharomyces / Q000201:enzymology; D017262:Siderophores / Q000096:biosynthesis; D012995:Solubility; D013379:Substrate Specificity; D013552:Swine
D002712:Chlorides; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010226:Parabens; D017262:Siderophores; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C042453:pyoverdin; D009952:Ornithine; D006899:Mixed Function Oxygenases; C524418:ornithine hydroxylase, Pseudomonas aeruginosa; C038193:4-hydroxybenzoic acid; D008239:Lysine