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Microbiology 154 (2008), 2804-2813; DOI  10.1099/mic.0.2008/018804-0
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Microbiology 154 (2008), 2804-2813; DOI  10.1099/mic.0.2008/018804-0
© 2008 Society for General Microbiology

Membrane-association determinants of the {omega}-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa

Francesco Imperi1,2,{dagger}, Lorenza Putignani2,{dagger},{ddagger}, Federica Tiburzi1,2, Cecilia Ambrosi1,§, Rita Cipollone1,||, Paolo Ascenzi1,2 and Paolo Visca1,2

1 Department of Biology, University ‘Roma Tre’, Viale G. Marconi 446, I-00146 Rome, Italy
2 National Institute for Infectious Diseases IRCCS, ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Rome, Italy

Correspondence
Paolo Visca
visca{at}uniroma3.it

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 {Delta}pvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.

Abbreviations: OHOrn, N{delta}-hydroxyornithine; Orn, L-ornithine; TM, transmembrane

{dagger}These authors contributed equally to this work.

{ddagger}Present address: Children's Hospital and Research Institute ‘Bambino Gesù’, Piazza S. Onofrio 4, I-00165 Rome, Italy.

§Present address: National Research Council, Istituto di Cristallografia, Sezione di Monterotondo, Monterotondo Stazione, I-00016 Rome, Italy.

||Present address: Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Via Montpellier 1, I-00133 Rome, Italy.

A multiple sequence alignment of PvdA is available with the online version of this paper.






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