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

Abstract

The L-ornithine N d -oxygenase PvdA catalyses the N d -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.

Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: Evidence for siderosomes

“…By contrast, cell fractionation experiments found the C-terminal PvdA fusion protein in both membrane and cytoplasmic fractions [43]. A similar distribution had been observed for wild-type PvdA [44] and is consistent with the presence at the N-terminus of the enzyme of a hydrophobic, inner-membrane-anchoring domain [24,44]. This it is a highly unstable compound [47] so must be promptly sequestered and used by the PVDI biosynthesis machinery once synthesized by PvdA and PvdF.…”

Repurposing the antimycotic drug flucytosine for suppression ofPseudomonas aeruginosapathogenicity

“…Pyochelin was detected by spraying with 0.1 M FeCl 3 and quantified by A 520 readings of ferripyochelin eluted with methanol from TLC plates (37). Anti-PvdA Western blot analysis was performed using the 3H6D12 monoclonal antibody as described (38).…”

Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa

“…E. coli was routinely used for recombinant DNA manipulations. Site-specific excision of pvdR and pvdA genes was performed as previously described (29). For complementation of pvdR, pvdT, and opmQ mutations, a 4.7-kb fragment encompassing the pvdRT-opmQ operon with its putative promoter (region 2642032-2646727 of the P. aeruginosa PAO1 chromosome, www.pseudomonas.com) was amplified by PCR and cloned in pUCP19, yielding plasmid pUCPpvdRTopmQ (see Table S1).…”