Heterologous expression, purification, and characterization of an l-ornithine N(5)-hydroxylase involved in pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa.
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.
Ge
Li
L
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Seah
Stephen Y K
SY
D021542:Amino Acids, Neutral / Q000378:metabolism / Q000494:pharmacology; D000613:Aminobutyrates / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006714:Homoserine / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000378:metabolism; D008239:Lysine / 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; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017262:Siderophores / Q000096:biosynthesis*; D013379:Substrate Specificity
D021542:Amino Acids, Neutral; D000613:Aminobutyrates; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; D017262:Siderophores; D005182:Flavin-Adenine Dinucleotide; D019811:Hydroxylamine; C005959:2,4-diaminobutyric acid; D009249:NADP; C013809:5-aminovaleric acid; D006714:Homoserine; C042453:pyoverdin; D009952:Ornithine; D006899:Mixed Function Oxygenases; D008239:Lysine