title: Membrane-association determinants of the omega-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa.
abstract: The L-ornithine N(delta)-oxygenase PvdA catalyses the N(delta)-hydroxylation of L-ornithine in many Pseudomonas spp., and thus provides an essential enzymic function in the biogenesis of the pyoverdine siderophore. Here, we report a detailed analysis of the membrane topology of the PvdA enzyme from the bacterial pathogen Pseudomonas aeruginosa. Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in Escherichia coli by constructing a series of translational fusions between PvdA and the PhoA (alkaline phosphatase) reporter enzyme. The inferred topological model resembled a eukaryotic reverse signal-anchor (type III) protein, with a single N-terminal domain anchored to the inner membrane, and the bulk of the protein spanning the cytosol. According to this model, the predicted transmembrane region should overlap the putative FAD-binding site. Cell fractionation and proteinase K accessibility experiments in P. aeruginosa confirmed the membrane-bound nature of PvdA, but excluded the transmembrane topology of its N-terminal hydrophobic region. Mutational analysis of PvdA, and complementation assays in a P. aeruginosa DeltapvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.
authors.affiliation: National Institute for Infectious Diseases IRCCS, 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Rome, Italy.|Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Imperi
authors.affiliation: National Institute for Infectious Diseases IRCCS, 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Rome, Italy.
authors.forename: Lorenza
authors.initials: L
authors.lastname: Putignani
authors.affiliation: National Institute for Infectious Diseases IRCCS, 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Rome, Italy.|Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Tiburzi
authors.affiliation: Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Ambrosi
authors.affiliation: Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Cipollone
authors.affiliation: National Institute for Infectious Diseases IRCCS, 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Rome, Italy.|Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Ascenzi
authors.affiliation: National Institute for Infectious Diseases IRCCS, 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Rome, Italy.|Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Visca