Membrane-association determinants of the omega-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa.
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.
Imperi
Francesco
F
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.
Putignani
Lorenza
L
National Institute for Infectious Diseases IRCCS, 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Rome, Italy.
Tiburzi
Federica
F
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.
Ambrosi
Cecilia
C
Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
Cipollone
Rita
R
Department of Biology, University 'Roma Tre', Viale G. Marconi 446, I-00146 Rome, Italy.
Ascenzi
Paolo
P
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.
Visca
Paolo
P
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.