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

Microbiology. 2008 Sep;154(Pt 9):2804-2813. doi: 10.1099/mic.0.2008/018804-0.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / chemistry
  • Alkaline Phosphatase / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Cloning, Molecular
  • DNA, Bacterial / genetics
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Deletion
  • Genes, Bacterial
  • Genetic Complementation Test
  • Hydrophobic and Hydrophilic Interactions
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / metabolism
  • Oligopeptides / biosynthesis
  • Ornithine / metabolism
  • Point Mutation
  • Protein Structure, Secondary
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Membrane Proteins
  • Oligopeptides
  • Recombinant Fusion Proteins
  • pyoverdin
  • Ornithine
  • Mixed Function Oxygenases
  • ornithine hydroxylase, Pseudomonas aeruginosa
  • Alkaline Phosphatase