Comprehensive Spectroscopic, Steady State, and Transient Kinetic Studies of a Representative Siderophore-associated Flavin Monooxygenase

• Published in: The Journal of biological chemistry Vol. 285; no. 40; pp. 30375 - 30388
• Main Authors: Mayfield, Jeffery A., Frederick, Rosanne E., Streit, Bennett R., Wencewicz, Timothy A., Ballou, David P., DuBois, Jennifer L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Antibiotics; Aspergillus fumigatus; Aspergillus fumigatus - enzymology; Enzymology; Flavin; Flavins - chemistry; Flavins - metabolism; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Fungi; Iron Metabolism; Kinetics; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Models, Chemical; Molecular Bases of Disease; Oxidase; Oxygen - chemistry; Oxygen - metabolism; Siderophores - chemistry; Siderophores - metabolism; Fungi; Flavin; Antibiotics; Iron Metabolism; Oxidase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.157578

Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread family of FAD-dependent monooxygenases. Here the kinetic mechanism for a representative family member has been extensively characterized by steady state and transient kinetic methods, using heterologously expressed N5-l-ornithine monooxygenase from the pathogenic fungus Aspergillus fumigatus. Spectroscopic data and kinetic analyses suggest a model in which a molecule of hydroxylatable substrate serves as an activator for the reaction of the reduced flavin and O2. The rate acceleration is only ∼5-fold, a mild effect of substrate on formation of the C4a-hydroperoxide that does not influence the overall rate of turnover. The effect is also observed with the bacterial ornithine monooxygenase PvdA. The C4a-hydroperoxide is stabilized in the absence of hydroxylatable substrate by the presence of bound NADP+ (t½ = 33 min, 25 °C, pH 8). NADP+ therefore is a likely regulator of O2 and substrate reactivity in the siderophore-associated monooxygenases. Aside from the activating effect of the hydroxylatable substrate, the siderophore-associated monooxygenases share a kinetic mechanism with the hepatic microsomal flavin monooxygenases and bacterial Baeyer-Villiger monooxygenases, with which they share only moderate sequence homology and from which they are distinguished by their acute substrate specificity. The remarkable specificity of the N5-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes.