title: The hFbpABC transporter from Haemophilus influenzae functions as a binding-protein-dependent ABC transporter with high specificity and affinity for ferric iron.
abstract: Pathogenic Haemophilus influenzae, Neisseria spp. (Neisseria gonorrhoeae and N. meningitidis), Serratia marcescens, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe3+ transport assay, we established an apparent Km=0.9 microM and Vmax=1.8 pmol/10(7)cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe3+ and selectivity for trivalent metals, including Ga3+ and Al3+, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli. Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity Kd=5.2 x 10(-4) M(-1), approximately 14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, approximately 35% of wild-type transport, with Km=1.2 microM and Vmax=0.5 pmol/10(7)cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe3+) binding and membrane transport.
authors.affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Room E1240 Biomedical Science Tower, Lothrop St., Pittsburgh, PA 15261, USA.
authors.forename: Damon S
authors.initials: DS
authors.lastname: Anderson
authors.forename: Pratima
authors.initials: P
authors.lastname: Adhikari
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Nowalk
authors.forename: Cheng Y
authors.initials: CY
authors.lastname: Chen
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Mietzner
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000502:physiology*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002412:Cations / Q000378:metabolism; D003300:Copper / Q000633:toxicity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D005708:Gallium / Q000633:toxicity; D005816:Genetic Complementation Test; D006193:Haemophilus influenzae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000032:analysis / Q000378:metabolism; D020125:Mutation, Missense; D009532:Nickel / Q000633:toxicity; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014475:Uncoupling Agents / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000633:toxicity
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002412:Cations; D004791:Enzyme Inhibitors; D005290:Ferric Compounds; D033902:Periplasmic Binding Proteins; D014475:Uncoupling Agents; D014638:Vanadates; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003300:Copper; D009532:Nickel; D005708:Gallium; D007501:Iron; D015032:Zinc