Molecular and sub-cellular topology of the enzyme L-ornithine N-oxygenase (PvdA) in Pseudomonas aeruginosa

Pyoverdine (PVD), the fluorescent siderophore of rRNA group I Pseudomonas spp., is composed of an invariant dihydroxyquinoline chromophore linked to a variable peptide chain. The peptide chain comprises some unusual amino acids, such as Nd-hydroxyornithine, Nd-formyl Nd-hydroxyornithine, b-hydroxyaspartate and b-hydroxyhistidine. Both peptide backbone and chromophore of PVDs are assembled by non-ribosomal peptide synthetases (NRPSs). In the complex scenario of PVD biogenesis an early reaction is driven by the L-ornithine Nd-oxygenase (PvdA), an w–aminoacid monooxygenase (EC 1.14.13.-) encoded by the pvdA gene, catalysing the Nd-hydroxylation of L-ornithine. We investigated the molecular and sub-cellular topology of PvdA from Pseudomonas aeruginosa PAO1. Membrane topogenic determinants of PvdA were identified by computational analysis and exploited to construct a series of translational fusions with b-galactosidase (b-Gal) or alkaline phosphatase (PhoA). The inferred topological model suggests a single N-terminal transmembrane domain, anchored to the inner membrane and a long hydrophilic loop exposed to the cytosol. The model was corroborated by mapping PvdA in the cellular membrane compartment with anti-PvdA monoclonal antibodies (MAbs) in immunoelectron microscopy (IMEM) studies. Western blot analysis of membrane fractions confirmed PvdA location within the inner membrane. Binding to the membrane was confirmed by an in vitro transcription/translation assays and characterised as a FAD/NAD(P)H-assisted translocation process. We speculate that the membrane-associated state of PvdA would facilitate oxygen and NAD(P)H recruitment for the oxygenase reaction and, at the same time, provide an anchoring site for the pyoverdine biosynthetic multienzyme complex, rendering possible an export-coupled synthesis process.