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
2019-11-21
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vol. , part 9, pp. 2804-2813

Multiple sequence alignment of PvdA with proximate homologues generated by the Jpred software ( http://barton.ebi.ac.uk/servers/jpred.html). Amino acid positions are indicated on top. Only regions containing conserved residues are considered by Jpred algorithms. Identical and similar residues common to the 22 aligned proteins are shaded in dark and light yellow, respectively. Similar residues are I, L, V and M; F and Y; D and E; S and T; R and K. Light green rectangles indicate the FAD, NAD(P)H and F/LATGY domains with strictly consensus motifs highlighted by black boxes. Protein sequences, with the accession number in parentheses, are: CDC1551 probable monooxygenase, Mon. (O53294); ATCC 19718 FAD-dependent pyridine nucleotide-disulphide oxidoreductase, Mon. (Q82TE8); ATCC 19089 uncharacterized monooxygenase, Mon. (Q9A588); uncharacterized flavin monooxygenase, FMO (Q802T5); uncharacterized flavin-containing monooxygenase, FMO3 (Q8QH01); ATCC 33970 uncharacterized oxidoreductase, Oxi. (Q8UJQ2); dimethylaniline monooxygenase, FMO2 (Q28505); monooxygenase, Mon. (O64489); l-Orn -oxygenase, Sid1 (P56584); monooxygenase, Mon. (Q9P7T0); hydroxylase, Hydro. (O94115); alcaligin biosynthesis enzyme, AlcA (P59855); monooxygenase, HxyA (Q9HHV0); l-lysine -hydroxylase, IucD (P11295); rhizobactin biosynthesis enzyme, RhbE (Q9Z3Q8); CO92 putative siderophore biosynthetic enzyme, AlcA (Q8ZFZ3); siderophore biosynthesis monooxygenase, Mon. (Q8ECU3); Orn -oxygenase, Mon. (Q8J2V1); uncharacterized monooxygenase, Mon. (Q9K9M5); putative monooxygenase, Mon. (Q9L071); sp. uncharacterized monooxygenase, Mon. sp. (Q8YZR5); PAO1 l-Orn -oxygenase, PvdA (Q51548). The predicted buried state (B) of PvdA amino acids with less than 5 % solvent accessibility is shown below the PvdA sequence. The inferred α-helices (H) and β-strands (E) of the PvdA consensus secondary structure are represented as red cylinders and green arrows, respectively. Negatively (D and E) and positively (R and K) charged PvdA amino acid residues are indicated by blue and red bold characters, respectively.



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