Kinetic Mechanism of Ornithine Hydroxylase (PvdA) from Pseudomonas aeruginosa: Substrate Triggering of O₂ Addition but Not Flavin Reduction

Abstract

PvdA catalyzes the hydroxylation of the sidechain primary amine of ornithine in the initial step of the biosynthesis of the Pseudomonas aeruginosa siderophore pyoverdin. The reaction requires FAD, NADPH, and O2. PvdA uses the same co-substrates as several flavin-dependent hydroxylases that differ one from another in the kinetic mechanisms of their oxidative and reductive half-reactions. Therefore, the mechanism of PvdA was determined by absorption stopped-flow experiments. By contrast to some flavin-dependent hydroxylases (notably, p-hydroxybenzoate hydroxylase), binding of the hydroxylation target is not required to trigger reduction of the flavin by NADPH: the reductive half-reaction is equally facile in the presence and absence of ornithine. Reaction of O2 with FADH2 in the oxidative half-reaction is accelerated by ornithine 80-fold, providing a mechanism by which PvdA can ensure coupling of NADPH and ornithine oxidation. In the presence of ornithine, the expected C(4a)-hydroperoxyflavin intermediate with 390-nm absorption accumulates and decays to the C(4a)-hydroxyflavin in a kinetically competent fashion. The slower oxidative half-reaction that occurs in the absence of ornithine involves accumulation of an oxygenated flavin species and two subsequent states that are tentatively assigned as C(4a)-peroxy- and -hydroperoxyflavin intermediates and the oxidized flavin. The enzyme generates stoichiometric hydrogen peroxide in lieu of hydroxyornithine. The data suggest that PvdA employs a kinetic mechanism that is a hybrid of those previously documented for other flavin-dependent hydroxylases.

Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus fumigatus Flavin-dependent Ornithine N5-Monooxygenase SidA

“…Thus, NADPH reduces the flavin, whereas NADP ϩ plays an essential role in the stabilization of flavin intermediates in SidA. This dual or moonlighting role of NADP(H) has also been observed in the reaction of the related ornithine hydroxylase PvdA from Pseudomonas aeruginosa (31)(32)(33). A moonlighting role for NADP(H) was originally proposed for other members of the Class B flavin-dependent monooxygenases (12,15,34).…”

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

“…The oxidation reaction of reduced enzyme-3HB, prepared either by NADH or dithionite reduction, showed similar kinetics. For Class B flavoprotein monooxygenases such as phenylacetone monooxygenase (31), cyclohexanone monooxgenase (32), and L-ornithine monooxygenase (33)(34)(35), the binding of NAD(P) ϩ is required to stabilize the C4a-peroxyflavin intermediate (36,37). For Class C enzymes such as bacterial luciferase (38,39) and Class D enzymes such as the oxygenase component of p-hydroxyphenylacetate hydroxylase (27,40), C4a-hydroperoxyflavin can be detected in the absence of any bound ligand.…”

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

“…These acids act as bidentate chelating moieties in the siderophores used by hundreds of organisms (27). Although these enzymes are now known to be widespread and appear to constitute a distinct subclass of flavin monooxygenases (28), they have undergone little mechanistic study until recently (35,42). Their encoding genes are essential for survival and/or virulence in many species of pathogenic fungi, notably deadly strains of A. fumigatus and intracellular Histoplasma capsulatum (15)(16)(17).…”

“…Transient kinetic and product inhibition data subsequently shed light on these results. First, as in PvdA, it was noted that the rate of formation of the C4a-hydroperoxide is regulated by the presence of substrate (42). The effect is mild, however, particularly when compared with the 10 5 -fold enhancement in the rate of FAD reduction in the presence of substrate observed for para-hydroxybenzoate hydroxylase (62).…”

“…Prior work with a bacterial OMO (PvdA) by Lamb and coworkers (35,42) led to the conclusion that PvdA might have mechanistic differences from the cohort of known FAD-dependent monooxygenases. This report provides the most detailed kinetic mechanistic studies, including both steady state and transient kinetic data, for any enzyme in the widespread SMO family.…”