Sub-cellular and molecular topology of L-ornithine Nd- oxygenase (PvdA) in Pseudomonas aeruginosa PAO1

Pyoverdines (PVDs), the fluorescent siderophores of rRNA group I Pseudomonas spp., are composed of an invariant dihydroxyquinoline chromophore linked to a variable peptide chain. Both the chromophore and the peptide backbone are assembled by non-ribosomal peptide synthetases (NRPSs). The peptide chain of Pseudomonas aeruginosa PAO1 PVD results from the condensation and partial cyclization of eight amino acids. In the complex scenario of PVDPAO1 biogenesis, a primary precursor-generating reaction is driven by 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. Membrane topogenic determinants of PvdA were identified by computational analysis and exploited to construct a series of translational fusions with 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 membrane compartment with anti-PvdA monoclonal antibodies (MAbs) in immunoelectron microscopy 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 assay and characterised as a FAD/NAD(P)H-assisted process. We speculate that the membrane-associated state of PvdA would facilitate oxygen and NAD(P)H recruitment for the oxygenase reaction while providing an anchoring site for the pyoverdine biosynthetic multienzyme complex, likely facilitating an export-coupled biosynthetic process