1887

Abstract

The -ornithine -oxygenase PvdA catalyses the -hydroxylation of -ornithine in many 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 . Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in 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 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 Δ mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.

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2008-09-01
2020-08-12
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