Communication between the Zinc and Nickel Sites in Dimeric HypA: Metal Recognition and pH Sensing.
Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site...
| Publicado en: | Journal of the American Chemical Society Vol. 132; no. 30; pp. 10338 - 10352 |
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| Autores principales: | , , , , , , , |
| Formato: | Artículo |
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American Chemical Society
8/4/2010
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| Materias: | |
| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=hlh&AN=52799898&site=ehost-live header: @attributes: shortDbName: hlh uiTerm: 52799898 longDbName: Humanities International Complete uiTag: AN controlInfo: bkinfo: jinfo: jid: 00027863 ACS jtl: Journal of the American Chemical Society issn: 00027863 maglogo: N pubinfo: dt: 8/4/2010 vid: 132 iid: 30 pid: 997 pub: American Chemical Society artinfo: ui: 52799898 10.1021/ja1005724 ppf: 10338 ppct: 14 formats: tig: atl: Communication between the Zinc and Nickel Sites in Dimeric HypA: Metal Recognition and pH Sensing. aug: au: Herbst, Robert W. Perovic, Iva Martin-Diaconescu, Vlad O'Brien, Kerrie Chivers, Peter T. Pochapsky, Susan Sondej Pochapsky, Thomas C. Maroney, Michael J. affil: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003 Departments of Chemistry and Biochemistry, Brandeis University, Waltham, Massachusetts 02454 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 su: Helicobacter pylori Hydrogen-ion concentration Zinc Nickel Metalloenzymes Nuclear magnetic resonance spectroscopy Hydrogenase sug: subj: Helicobacter pylori Hydrogen-ion concentration Zinc Nickel Metalloenzymes Nuclear magnetic resonance spectroscopy Hydrogenase ab: Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys) site to a Zn(His)(Cys) site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the β-CH protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys)) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the flow of nickel traffic in the cell in response to nickel availability and pH. pubtype: Academic Journal doctype: Article src: R language: English refInfo: copyright: @attributes: flag: Y dt: @attributes: year: 2010 holdings: @attributes: islocal: N |
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