title: Conserved residues and motifs in the NixA protein of Helicobacter pylori are critical for the high affinity transport of nickel ions.
abstract: NixA, the high affinity nickel transport protein of Helicobacter pylori, imports Ni2+ ions across the cytoplasmic membrane for insertion into the active site of the urease metalloenzyme, which is essential for colonization of the gastric mucosa. Twelve conserved aspartate (aspartates 47, 49, 55, 194, 231, and 234), glutamate (glutamates 106, 198, and 274), and histidine (histidines 44, 50, and 79) residues were identified by alignment of NixA with homologous transporters. Polymerase chain reaction-generated site-directed mutants of these residues were expressed in E. coli along with the H. pylori urease gene cluster. Mutations in residues within the predicted periplasmic domains of NixA maintained near wild type levels of Ni2+ uptake and urease activity, as did control mutations of conserved positively charged residues (lysines 140 and 268; arginines 162 and 167). Mutations in highly conserved motifs in predicted helices II and III of NixA abolished Ni2+ uptake and urease activity. Mutations in helices V and VI and the cytoplasmic domains decreased Ni2+ transport rates by >/=90%. Reduction in rates of Ni2+ transport correlated with reduction in urease activities (r = 0.77). Ni2+ transport was inhibited in the presence of Co2+, Cu2+, and Zn2+, indicating that these ions may also be bound or transported by NixA. We conclude that conserved Asp, Glu, and His residues in the transmembrane domains of NixA are critical for the transport of the divalent cations Ni2+, Co2+, Cu2+, and Zn2+ into the cytoplasm of H. pylori.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Fulkerson
authors.forename: R M
authors.initials: RM
authors.lastname: Garner
authors.forename: H L
authors.initials: HL
authors.lastname: Mobley
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D017124:Conserved Sequence; D016480:Helicobacter pylori / Q000378:metabolism*; D017136:Ion Transport; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism*; D014510:Urease / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D008565:Membrane Proteins; C095257:NixA protein, Helicobacter pylori; D009532:Nickel; D014510:Urease