Kinetic mechanism of ornithine hydroxylase (PvdA) from Pseudomonas aeruginosa: substrate triggering of O2 addition but not flavin reduction.

The opportunistic human pathogen Pseudomonas aeruginosa produces pyoverdin, a hydroxamate siderophore, to scavenge iron in the iron-limiting conditions of the host, a process that has been linked to virulence (1–3). Pyoverdin contains a chromophore and a peptide backbone of 8–12 amino acids, formed by nonribosomal peptide synthetase enzymes (NRPS) (2, 4, 5). The amino acids of the peptide backbone are both proteinogenic and non-proteinogenic and include two formyl-hydroxyornithines. These two unusual amino acids provide hydroxamate ligands for iron chelation. Two accessory enzymes to the NRPS, the ornithine hydroxylase (PvdA) (6, 7) and the hydroxyornithine transformylase (PvdF) (8, 9), prepare ornithine for incorporation into the siderophore by the NRPS assembly system. PvdA was recently characterized by steady-state kinetics and found to require both FAD and NADPH and to have high specificity for ornithine (Figure 1A) (10). The enzyme was tested for activity by two methods: the formation of hydroxylated product, which showed inhibition at high ornithine concentrations, and the oxidation of NADPH, which demonstrated simple saturation kinetics. PvdA was inhibited by chloride ions, and lysine served as a non-substrate effector, activating NADPH oxidation without the formation of hydroxylysine. It was noted that, in several respects, PvdA is functionally homologous to para-hydroxybenzoate hydroxylase (PHBH) and flavin-containing monooxygenase (FMO), two monooxygenases to which PvdA is comparably similar (∼ 35% at the level of primary structure). In both PHBH and FMO, addition of O2 to the reduced flavin generates a C(4a)-hydroperoxyflavin intermediate, which decays by substrate hydroxylation to the C(4a)-hydroxyflavin (11–15). This flavin intermediate sequence has been demonstrated by stopped-flow absorption and fluorescence experiments, in which both the hydroperoxy- and hydroxyflavins have absorbance maxima at 375–395 nm, whereas the hydroxyflavin is also highly fluorescent (excitation ∼400 nm and emission > 500 nm) (16–19). Despite the similarity of their chemical mechanisms, the two enzymes (PHBH and FMO) employ different kinetic mechanisms. PHBH requires the presence of substrate for the reduction of the flavin by NADPH (Figure 1B), which is hypothesized to be a regulatory mechanism to prevent uncoupled NADPH oxidation (12, 13, 18). In contrast, the flavin of FMO can be reduced by NADPH regardless of whether the target substrate is present (Figure 1C). The C(4a)-hydroperoxyflavin intermediate produced by subsequent addition of O2 to the reduced cofactor is remarkably stable (half-life ∼ 2 hrs) and awaits binding of the substrate. The protection of the intermediate ensures that, once NADPH has been oxidized, substrate oxidation ensues (11, 14, 18, 20). Thus, PHBH and FMO embody two distinct mechanistic strategies to ensure coupling of NADPH and substrate oxidation. Figure 1 Reaction catalyzed by (A) ornithine hydroxylase (PvdA) from P. aerugionsa, (B) p-hydroxybenzoate hydroxylase (PHBH) from P. fluorescens, and (C) flavin-containing monooxygenase (FMO) from hog liver microsomes. The substrate for PHBH (p-hydroxybenzoate ... The previous steady-state kinetic experiments on PvdA suggested that FAD reduction does not require ornithine. However, the upper limit that could be set for the half-life of the presumptive hydroxylating intermediate was much shorter than would seem to be required for PvdA to use the FMO coupling strategy (10). This work thus raised the question of whether PvdA ensures coupling of NADPH oxidation and ornithine hydroxylation, and how the enzyme might accomplish that coupling. Here, we have used stopped-flow absorption measurements to elucidate the PvdA mechanism. The results show that, whereas the enzyme follows the canonical chemical mechanism involving the key C(4a)-hydroperoxyflavin intermediate, PvdA coupling is regulated by a mechanism that is distinct from those employed by PHBH and FMO. This novel mechanism involves substrate triggering of O2 addition to the reduced flavin.