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

Francesco Imperi, Lorenzo Putignani, Federica Tiburzi, Cecilia Ambrosi, Rita Cipollone, Paolo Ascenzi, Paolo Visca

Research output: Contribution to journalArticle

Abstract

The L-ornithine Nδ-oxygenase PvdA catalyses the Nδ-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 DpvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.

Original languageEnglish
Pages (from-to)2804-2813
Number of pages10
JournalMicrobiology
Volume154
Issue number9
DOIs
Publication statusPublished - 2008

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Mixed Function Oxygenases
Pseudomonas aeruginosa
Ornithine
Amino Acids
Membranes
Enzymes
Cell Fractionation
Siderophores
Endopeptidase K
Oxygenases
Flavin-Adenine Dinucleotide
Hydroxylation
Pseudomonas
Cytosol
Alkaline Phosphatase
Membrane Proteins
Binding Sites
Escherichia coli
pyoverdin
Proteins

ASJC Scopus subject areas

  • Microbiology

Cite this

Membrane-association determinants of the ω-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa. / Imperi, Francesco; Putignani, Lorenzo; Tiburzi, Federica; Ambrosi, Cecilia; Cipollone, Rita; Ascenzi, Paolo; Visca, Paolo.

In: Microbiology, Vol. 154, No. 9, 2008, p. 2804-2813.

Research output: Contribution to journalArticle

Imperi, Francesco ; Putignani, Lorenzo ; Tiburzi, Federica ; Ambrosi, Cecilia ; Cipollone, Rita ; Ascenzi, Paolo ; Visca, Paolo. / Membrane-association determinants of the ω-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa. In: Microbiology. 2008 ; Vol. 154, No. 9. pp. 2804-2813.
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