Identification of residues of FpvA involved in the different steps of Pvd-Fe uptake in Pseudomonas aeruginosa. - Archive ouverte HAL Access content directly
Journal Articles Biochemistry Year : 2007

Identification of residues of FpvA involved in the different steps of Pvd-Fe uptake in Pseudomonas aeruginosa.

Abstract

FpvA is an outer membrane transporter involved in iron uptake by the siderophore pyoverdine (Pvd) in Pseudomonas aeruginosa. This transporter, like all other proteins of the same family, consists of a transmembrane 22 beta-stranded barrel occluded by a plug domain. The beta-strands of the barrel are connected by large extracellular loops and short periplasmic turns. Site-directed mutagenesis was carried out on FpvA to identify the extracellular loops or parts of these loops involved in the various stages of Pvd-Fe uptake. The G286C, W362C, and W434C mutations in loops L1, L3, and L4, respectively, disturbed the binding of the apo siderophore, as shown by time-resolved fluorescence spectroscopy. Iron uptake experiments followed by fluorescence resonance energy transfer (FRET) or using 55Fe indicated that residues W434 and G701 and, therefore, loops L4 and L9 must be involved in Pvd-Fe uptake by FpvA. The two corresponding mutants incorporated smaller than normal amounts of 55Fe into cells, and no Pvd recycling on FpvA was observed after iron release. Surprisingly, the S603C mutation in loop L7 increased the amount of Pvd-Fe transported. Our results suggest that W434 (L4), S603 (L7), and G701 (L9) are involved in the mechanism of Pvd-Fe uptake.

Dates and versions

hal-00344057 , version 1 (03-12-2008)

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Mirella Nader, Wim Dobbelaere, Michel Vincent, Laure Journet, Hendrik Adams, et al.. Identification of residues of FpvA involved in the different steps of Pvd-Fe uptake in Pseudomonas aeruginosa.. Biochemistry, 2007, 46 (42), pp.11707-17. ⟨10.1021/bi700997w⟩. ⟨hal-00344057⟩
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