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 language | English |
|---|---|
| Pages (from-to) | 2804-2813 |
| Number of pages | 10 |
| Journal | Microbiology |
| Volume | 154 |
| Issue number | 9 |
| DOIs | |
| Publication status | Published - 2008 |
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- Microbiology
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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 journal › Article
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TY - JOUR
T1 - Membrane-association determinants of the ω-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa
AU - Imperi, Francesco
AU - Putignani, Lorenzo
AU - Tiburzi, Federica
AU - Ambrosi, Cecilia
AU - Cipollone, Rita
AU - Ascenzi, Paolo
AU - Visca, Paolo
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=53449085207&partnerID=8YFLogxK
U2 - 10.1099/mic.0.2008/018804-0
DO - 10.1099/mic.0.2008/018804-0
M3 - Article
C2 - 18757814
AN - SCOPUS:53449085207
VL - 154
SP - 2804
EP - 2813
JO - Microbiology
JF - Microbiology
SN - 1350-0872
IS - 9
ER -