title: Comprehensive spectroscopic, steady state, and transient kinetic studies of a representative siderophore-associated flavin monooxygenase.
abstract: 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 N(5)-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 O(2). 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(+) (t(½) = 33 min, 25 °C, pH 8). NADP(+) therefore is a likely regulator of O(2) 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 N(5)-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Jeffery A
authors.initials: JA
authors.lastname: Mayfield
authors.forename: Rosanne E
authors.initials: RE
authors.lastname: Frederick
authors.forename: Bennett R
authors.initials: BR
authors.lastname: Streit
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Wencewicz
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: DuBois
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology*; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017262:Siderophores / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005415:Flavins; D005656:Fungal Proteins; D017262:Siderophores; D006899:Mixed Function Oxygenases; C059419:ornithine N5-oxygenase; C524418:ornithine hydroxylase, Pseudomonas aeruginosa; D010100:Oxygen