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1
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Benoit SL, Maier RJ. Copper toxicity towards Campylobacter jejuni is enhanced by the nickel chelator dimethylglyoxime. Metallomics 2021; 14:6486457. [PMID: 34963007 DOI: 10.1093/mtomcs/mfab076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Accepted: 12/20/2021] [Indexed: 11/13/2022]
Abstract
The nickel (Ni)-chelator dimethylglyoxime (DMG) was found to be bacteriostatic towards Campylobacter jejuni. Supplementation of nickel to DMG-containing media restored bacterial growth, whereas supplementation of cobalt or zinc had no effect on the growth inhibition. Unexpectedly, the combination of millimolar levels of DMG with micromolar levels of copper (Cu) was bactericidal, an effect not seen in select Gram-negative pathogenic bacteria. Both the cytoplasmic Ni-binding chaperone SlyD and the twin arginine translocation (Tat)-dependent periplasmic copper oxidase CueO were found to play a central role in the Cu-DMG hypersensitivity phenotype. Ni-replete SlyD is needed for Tat-dependent CueO translocation to the periplasm, whereas Ni-depleted (DMG-treated) SlyD is unable to interact with the CueO Tat signal peptide, leading to mislocalization of CueO and increased copper sensitivity. In support of this model, C. jejuni ΔslyD and ΔcueO mutants were more sensitive to copper than the wild-type (WT); CueO was less abundant in the periplasmic fraction of ΔslyD or DMG-grown WT cells, compared to WT cells grown on plain medium; SlyD binds the CueO signal sequence peptide, with DMG inhibiting and nickel enhancing the binding, respectively. Injection of Cu-DMG into Galleria mellonella before C. jejuni inoculation significantly increased the insect survival rate compared to the control group. In chickens, oral administration of DMG or Cu-DMG decreased and even abolished C. jejuni colonization in some cases, compared to both water-only and Cu-only control groups. The latter finding is important, since campylobacteriosis is the leading bacterial foodborne infection, and chicken meat constitutes the major foodborne source.
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Affiliation(s)
- Stéphane L Benoit
- Department of Microbiology.,Center for Metalloenzyme Studies, The University of Georgia, Athens, Georgia, 30602
| | - Robert J Maier
- Department of Microbiology.,Center for Metalloenzyme Studies, The University of Georgia, Athens, Georgia, 30602
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2
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Benoit SL, Agudelo S, Maier RJ. A two-hybrid system reveals previously uncharacterized protein-protein interactions within the Helicobacter pylori NIF iron-sulfur maturation system. Sci Rep 2021; 11:10794. [PMID: 34031459 PMCID: PMC8144621 DOI: 10.1038/s41598-021-90003-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2021] [Accepted: 04/26/2021] [Indexed: 11/10/2022] Open
Abstract
Iron-sulfur (Fe-S) proteins play essential roles in all living organisms. The gastric pathogen Helicobacter pylori relies exclusively on the NIF system for biosynthesis and delivery of Fe-S clusters. Previously characterized components include two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein), and a dispensable Fe-S carrier, Nfu. Among 38 proteins previously predicted to coordinate Fe-S clusters, two proteins, HP0207 (a member of the Nbp35/ApbC ATPase family) and HP0277 (previously annotated as FdxA, a member of the YfhL ferredoxin-like family) were further studied, using a bacterial two-hybrid system approach to identify protein-protein interactions. ApbC was found to interact with 30 proteins, including itself, NifS, NifU, Nfu and FdxA, and alteration of the conserved ATPase motif in ApbC resulted in a significant (50%) decrease in the number of protein interactions, suggesting the ATpase activity is needed for some ApbC-target protein interactions. FdxA was shown to interact with 21 proteins, including itself, NifS, ApbC and Nfu, however no interactions between NifU and FdxA were detected. By use of cross-linking studies, a 51-kDa ApbC-Nfu heterodimer complex was identified. Attempts to generate apbC chromosomal deletion mutants in H. pylori were unsuccessful, therefore indirectly suggesting the hp0207 gene is essential. In contrast, mutants in the fdxA gene were obtained, albeit only in one parental strain (26695). Taken together, these results suggest both ApbC and FdxA are important players in the H. pylori NIF maturation system.
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Affiliation(s)
- Stéphane L Benoit
- Department of Microbiology, The University of Georgia, 30602, Athens, Georgia.,Center for Metalloenzyme Studies, The University of Georgia, 30602, Athens, Georgia
| | - Stephanie Agudelo
- Department of Microbiology, The University of Georgia, 30602, Athens, Georgia
| | - Robert J Maier
- Department of Microbiology, The University of Georgia, 30602, Athens, Georgia. .,Center for Metalloenzyme Studies, The University of Georgia, 30602, Athens, Georgia.
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3
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Liu Y, Yuan B, Peng L, Zhao J, Cheng B, Huang Y, Zheng X, Zhou Y, Xiang S, Zhu L, Wu Y. Single-particle analysis of urea amidolyase reveals its molecular mechanism. Protein Sci 2020; 29:1242-1249. [PMID: 32105377 DOI: 10.1002/pro.3847] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2019] [Revised: 02/17/2020] [Accepted: 02/17/2020] [Indexed: 11/08/2022]
Abstract
Urea amidolyase (UA), a bifunctional enzyme that is widely distributed in bacteria, fungi, algae, and plants, plays a pivotal role in the recycling of nitrogen in the biosphere. Its substrate urea is ultimately converted to ammonium, via successive catalysis at the C-terminal urea carboxylase (UC) domain and followed by the N-terminal allophanate hydrolyse (AH) domain. Although our previous studies have shown that Kluyveromyces lactis UA (KlUA) functions efficiently as a homodimer, the architecture of the full-length enzyme remains unresolved. Thus how the biotin carboxyl carrier protein (BCCP) domain is transferred within the UC domain remains unclear. Here we report the structures of full-length KlUA in its homodimer form in three different functional states by negatively-stained single-particle electron microscopy. We report here that the ADP-bound structure with or without urea shows two possible locations of BCCP with preferred asymmetry, and that when BCCP is attached to the carboxyl transferase domain of one monomer, it is attached to the biotin carboxylase domain in the second domain. Based on this observation, we propose a BCCP-swinging model for biotin-dependent carboxylation mechanism of this enzyme.
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Affiliation(s)
- Ying Liu
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Bin Yuan
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Liang Peng
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Jing Zhao
- Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Medical University, Tianjin, China
| | - Bin Cheng
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Yuhua Huang
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Xinxing Zheng
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Yuerong Zhou
- College of Marine and Biochemical Engineering, Fujian Normal University, Fuzhou, China
| | - Song Xiang
- Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Medical University, Tianjin, China
| | - Li Zhu
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Yi Wu
- MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China
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4
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Zambelli B, Mazzei L, Ciurli S. Intrinsic disorder in the nickel-dependent urease network. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2020; 174:307-330. [DOI: 10.1016/bs.pmbts.2020.05.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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Abstract
Nickel is essential for the survival of many pathogenic bacteria. E. coli and H. pylori require nickel for [NiFe]-hydrogenases. H. pylori also requires nickel for urease. At high concentrations nickel can be toxic to the cell, therefore, nickel concentrations are tightly regulated. Metalloregulators help to maintain nickel concentration in the cell by regulating the expression of the genes associated with nickel import and export. Nickel import into the cell, delivery of nickel to target proteins, and export of nickel from the cell is a very intricate and well-choreographed process. The delivery of nickel to [NiFe]-hydrogenase and urease is complex and involves several chaperones and accessory proteins. A combination of biochemical, crystallographic, and spectroscopic techniques has been utilized to study the structures of these proteins, as well as protein-protein interactions resulting in an expansion of our knowledge regarding how these proteins sense and bind nickel. In this review, recent advances in the field will be discussed, focusing on the metal site structures of nickel bound to metalloregulators and chaperones.
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6
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Noncatalytic Antioxidant Role for Helicobacter pylori Urease. J Bacteriol 2018; 200:JB.00124-18. [PMID: 29866802 DOI: 10.1128/jb.00124-18] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Accepted: 05/30/2018] [Indexed: 12/15/2022] Open
Abstract
The well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protect Helicobacter pylori against the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ΔureAB deletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protect H. pylori against oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purified H. pylori urease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.IMPORTANCE Chronic Helicobacter pylori infection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid, H. pylori must survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge of H. pylori survival mechanisms.
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Saylor Z, Maier R. Helicobacter pylori nickel storage proteins: recognition and modulation of diverse metabolic targets. Microbiology (Reading) 2018; 164:1059-1068. [DOI: 10.1099/mic.0.000680] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Affiliation(s)
- Zachary Saylor
- Department of Microbiology and Center for Metalloprotein Studies, University of Georgia, Athens, GA, USA
| | - Robert Maier
- Department of Microbiology and Center for Metalloprotein Studies, University of Georgia, Athens, GA, USA
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Hu HQ, Huang HT, Maroney MJ. The Helicobacter pylori HypA·UreE 2 Complex Contains a Novel High-Affinity Ni(II)-Binding Site. Biochemistry 2018; 57:2932-2942. [PMID: 29708738 DOI: 10.1021/acs.biochem.8b00127] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Helicobacter pylori is a human pathogen that colonizes the stomach, is the major cause of ulcers, and has been associated with stomach cancers. To survive in the acidic environment of the stomach, H. pylori uses urease, a nickel-dependent enzyme, to produce ammonia for maintenance of cellular pH. The bacteria produce apo-urease in large quantities and activate it by incorporating nickel under acid shock conditions. Urease nickel incorporation requires the urease-specific metallochaperone UreE and the (UreFGH)2 maturation complex. In addition, the H. pylori nickel urease maturation pathway recruits accessory proteins from the [NiFe] hydrogenase maturation pathway, namely, HypA and HypB. HypA and UreE dimers (UreE2) are known to form a protein complex, the role of which in urease maturation is largely unknown. Herein, we examine the nickel-binding properties and protein-protein interactions of HypA and UreE2 using isothermal titration calorimetry and fluorometric methods under neutral and acidic pH conditions to gain insight into the roles played by HypA in urease maturation. The results reveal that HypA and UreE2 form a stable complex with micromolar affinity that protects UreE from hydrolytic degradation. The HypA·UreE2 complex contains a unique high-affinity (nanomolar) Ni2+-binding site that is maintained under conditions designed to mimic acid shock (pH 6.3). The data are interpreted in terms of a proposed mechanism wherein HypA and UreE2 act as co-metallochaperones that target the delivery of Ni2+ to apo-urease with high fidelity.
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Benoit SL, Holland AA, Johnson MK, Maier RJ. Iron-sulfur protein maturation in Helicobacter pylori: identifying a Nfu-type cluster carrier protein and its iron-sulfur protein targets. Mol Microbiol 2018; 108:379-396. [PMID: 29498770 DOI: 10.1111/mmi.13942] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/23/2018] [Indexed: 01/03/2023]
Abstract
Helicobacter pylori is anomalous among non nitrogen-fixing bacteria in containing an incomplete NIF system for Fe-S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU-depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild-type (WT) strains. The hp1492 gene, encoding a putative Nfu-type Fe-S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe-2S] or [4Fe-4S] cluster/dimer, based on analytical, UV-visible absorption/CD and resonance Raman studies. A bacterial two-hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe-S-containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe-S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe-S clusters in H. pylori.
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Affiliation(s)
- Stéphane L Benoit
- Department of Microbiology and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA
| | - Ashley A Holland
- Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA
| | - Michael K Johnson
- Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA
| | - Robert J Maier
- Department of Microbiology and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA
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10
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Tepeš B, Malfertheiner P, Labenz J, Aygen S. Modified Helicobacter test using a new test meal and a 13C-urea breath test in Helicobacter pylori positive and negative dyspepsia patients on proton pump inhibitors. World J Gastroenterol 2017; 23:5954-5961. [PMID: 28932087 PMCID: PMC5583580 DOI: 10.3748/wjg.v23.i32.5954] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Revised: 03/25/2017] [Accepted: 06/01/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To determine the sensitivity and specificity of the 13C-urea breath test (UBT) in patients taking proton pump inhibitors (PPIs), using a new test meal Refex.
METHODS One hundred and fourteen consecutive patients with dyspepsia, 53 Helicobacter pylori (H. pylori) positive, 49 H. pylori negative, were included in the study. The patients were then given esomeprazole 40 mg for 29 consecutive days, and the 13C-UBT with the new test meal was performed the next morning.
RESULTS The sensitivity of the 13C-UBT with a cut off 2.5‰ was 92.45% (95%CI: 81.79%-97.91%) by per-protocol (PP) analysis and 78.13% (95%CI: 66.03%-87.49%) by intention-to-treat (ITT) analysis. The specificity of the 13C-UBT test was 96.00% in the ITT population (95%CI: 86.29%-99.51%) and 97.96% in the PP population (95%CI: 89.15%-99.95%).
CONCLUSION The new test meal based 13C-UBT is highly accurate in patients on PPIs and can be used in those unable to stop their PPI treatment.
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Affiliation(s)
- Bojan Tepeš
- AM DC Rogaška, Prvomajska 29 A, 3250 Rogaška Slatina, Slovenia
| | - Peter Malfertheiner
- Universitätsklinikum Magdeburg A. ö. R Klinik für Gastroenterologie, Hepatologie und Infektiologie, 39120 Magdeburg, Germany
| | - Joachim Labenz
- Department of Internal Medicine and Gastroenterology, Diakonie Klinikum, Jung-Stilling Hospital, 57074 Siegen, Germany
| | - Sitke Aygen
- Institut für Biomedizinische Analytik und NMR-Imaging GmbH (INFAI), Gottfried-Hagen-Str. 6062, 51105 Köln, Germany
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11
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Blum FC, Hu HQ, Servetas SL, Benoit SL, Maier RJ, Maroney MJ, Merrell DS. Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori. PLoS One 2017; 12:e0183260. [PMID: 28809946 PMCID: PMC5557546 DOI: 10.1371/journal.pone.0183260] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2017] [Accepted: 08/01/2017] [Indexed: 01/18/2023] Open
Abstract
The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.
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Affiliation(s)
- Faith C. Blum
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America
| | - Heidi Q. Hu
- Department of Chemistry and Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA, United States of America
| | - Stephanie L. Servetas
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America
| | - Stéphane L. Benoit
- Department of Microbiology, University of Georgia, Athens, GA, United States of America
| | - Robert J. Maier
- Department of Microbiology, University of Georgia, Athens, GA, United States of America
| | - Michael J. Maroney
- Department of Chemistry and Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA, United States of America
- * E-mail: (MJM); (DSM)
| | - D. Scott Merrell
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America
- * E-mail: (MJM); (DSM)
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12
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Nakashige TG, Zygiel EM, Drennan CL, Nolan EM. Nickel Sequestration by the Host-Defense Protein Human Calprotectin. J Am Chem Soc 2017; 139:8828-8836. [PMID: 28573847 PMCID: PMC5754018 DOI: 10.1021/jacs.7b01212] [Citation(s) in RCA: 92] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The human innate immune protein calprotectin (CP, S100A8/S100A9 oligomer, calgranulin A/calgranulin B oligomer, MRP-8/MRP-14 oligomer) chelates a number of first-row transition metals, including Mn(II), Fe(II), and Zn(II), and can withhold these essential nutrients from microbes. Here we elucidate the Ni(II) coordination chemistry of human CP. We present a 2.6-Å crystal structure of Ni(II)- and Ca(II)-bound CP, which reveals that CP binds Ni(II) ions at both its transition-metal-binding sites: the His3Asp motif (site 1) and the His6 motif (site 2). Further biochemical studies establish that coordination of Ni(II) at the hexahistidine site is thermodynamically preferred over Zn(II). We also demonstrate that CP can sequester Ni(II) from two human pathogens, Staphylococcus aureus and Klebsiella pneumoniae, that utilize this metal nutrient during infection, and inhibit the activity of the Ni(II)-dependent enzyme urease in bacterial cultures. In total, our findings expand the biological coordination chemistry of Ni(II)-chelating proteins in nature and provide a foundation for evaluating putative roles of CP in Ni(II) homeostasis at the host-microbe interface and beyond.
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Affiliation(s)
- Toshiki G. Nakashige
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Emily M. Zygiel
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Catherine L. Drennan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Elizabeth M. Nolan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
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13
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Hu HQ, Johnson RC, Merrell DS, Maroney MJ. Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori. Biochemistry 2017; 56:1105-1116. [PMID: 28177601 DOI: 10.1021/acs.biochem.6b00912] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.
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Affiliation(s)
- Heidi Q Hu
- Department of Chemistry and Program of Molecular and Cellular Biology, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States
| | - Ryan C Johnson
- Microbiology and Immunology, Uniformed Services University of the Health Sciences , Bethesda, Maryland 20814, United States
| | - D Scott Merrell
- Microbiology and Immunology, Uniformed Services University of the Health Sciences , Bethesda, Maryland 20814, United States
| | - Michael J Maroney
- Department of Chemistry and Program of Molecular and Cellular Biology, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States
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14
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Vinella D, Fischer F, Vorontsov E, Gallaud J, Malosse C, Michel V, Cavazza C, Robbe-Saule M, Richaud P, Chamot-Rooke J, Brochier-Armanet C, De Reuse H. Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization. PLoS Pathog 2015; 11:e1005312. [PMID: 26641249 PMCID: PMC4671568 DOI: 10.1371/journal.ppat.1005312] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2015] [Accepted: 11/05/2015] [Indexed: 02/07/2023] Open
Abstract
Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori. Helicobacter pylori is a bacterium that persistently colonizes the stomach of half of the human population. Infection by H. pylori is associated with gastritis, peptic ulcer disease and adenocarcinoma. To resist gastric acidity and proliferate in the stomach, H. pylori relies on urease, an enzyme that contains a nickel-metallocenter at its active site. Thus, nickel is a virulence determinant for H. pylori. Our aim is to characterize how H. pylori controls the intracellular nickel concentration to avoid toxicity, which protein partners are involved, and how they impact urease activity and virulence. We characterized two H. pylori proteins, Hpn and Hpn-2 that are rich in Histidine residues. We demonstrated that Hpn is involved in nickel sequestration, that the two proteins interact with each other and that their combined activities participate in a nickel transfer pathway to urease. Hpn is only expressed in gastric Helicobacter species able to colonize the stomach and Hpn-2 is restricted to the H. pylori and its close relative H. acinonychis. We found that both proteins are essential for colonization of a mouse model by H. pylori. We conclude that during evolution, the acquisition of Hpn and Hpn-2 by gastric Helicobacter species was decisive for their capacity to colonize the stomach.
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Affiliation(s)
- Daniel Vinella
- Institut Pasteur, Département de Microbiologie, Unité Pathogenèse de Helicobacter, ERL CNRS 3526, Paris, France
| | - Frédéric Fischer
- Institut Pasteur, Département de Microbiologie, Unité Pathogenèse de Helicobacter, ERL CNRS 3526, Paris, France
| | - Egor Vorontsov
- Institut Pasteur, Département de Biologie Structurale et Chimie, Unité Spectrométrie de Masse Structurale et Protéomique, CNRS UMR 3528, Paris, France
| | - Julien Gallaud
- Institut Pasteur, Département de Microbiologie, Unité Pathogenèse de Helicobacter, ERL CNRS 3526, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France
| | - Christian Malosse
- Institut Pasteur, Département de Biologie Structurale et Chimie, Unité Spectrométrie de Masse Structurale et Protéomique, CNRS UMR 3528, Paris, France
| | - Valérie Michel
- Institut Pasteur, Département de Microbiologie, Unité Pathogenèse de Helicobacter, ERL CNRS 3526, Paris, France
| | | | - Marie Robbe-Saule
- Institut Pasteur, Département de Microbiologie, Unité Pathogenèse de Helicobacter, ERL CNRS 3526, Paris, France
| | - Pierre Richaud
- CEA, DSV, IBEB, SBVME and CNRS, UMR 7265 Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance, France and Aix Marseille Université, BVME UMR7265, Marseille, France
| | - Julia Chamot-Rooke
- Institut Pasteur, Département de Biologie Structurale et Chimie, Unité Spectrométrie de Masse Structurale et Protéomique, CNRS UMR 3528, Paris, France
| | - Céline Brochier-Armanet
- Université de Lyon, Université Lyon 1, CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, Villeurbanne, France
| | - Hilde De Reuse
- Institut Pasteur, Département de Microbiologie, Unité Pathogenèse de Helicobacter, ERL CNRS 3526, Paris, France
- * E-mail:
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15
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Yang X, Li H, Cheng T, Xia W, Lai YT, Sun H. Nickel translocation between metallochaperones HypA and UreE in Helicobacter pylori. Metallomics 2015; 6:1731-6. [PMID: 25010720 DOI: 10.1039/c4mt00134f] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Incorporation of nickel ions to the active sites of urease and hydrogenase is prerequisite for the appropriate functions of the metalloenzymes. Such a process requires the participation of several accessory proteins. Interestingly, some of them are shared by the two enzymes in their maturation processes. In this work, we characterized the molecular details of the interaction of metallochaperones UreE and HypA in Helicobacter pylori. We show by chemical cross-linking and static light scattering that the UreE dimer binds to HypA to form a hetero-complex i.e. HypA-(UreE)2. The dissociation constant (Kd) of the protein complex was determined by ITC to be 1 μM in the absence of nickel ions; whereas binding of Ni(2+) but not Zn(2+) to UreE resulted in ca. one fold decrease in the affinity. The putative interfaces on HypA unveiled by NMR chemical shift perturbation were found mainly at the nickel binding domain and in the cleft between α1 and β1/β6. We also identified that the C-domain of UreE, in particular the C-terminal residues of 158-170 are indispensable for the interaction of UreE and HypA. Such an interaction was also observed intracellularly by GFP-fragment reassembly assay. Moreover, we demonstrated using a fluorescent probe that nickel is transferred from HypA to UreE via the specific protein-protein interaction. Deletion of the C-terminus (residues 158-170) of UreE abolished nickel transfer and led to a significant decrease in urease activity. This study provides direct in vitro and in vivo evidence as well as molecular details of nickel translocation mediated by protein-protein interaction.
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Affiliation(s)
- Xinming Yang
- Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P.R. China.
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16
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Rowinska-Zyrek M, Zakrzewska-Czerwinska J, Zawilak-Pawlik A, Kozlowski H. Ni²⁺ chemistry in pathogens--a possible target for eradication. Dalton Trans 2014; 43:8976-8989. [PMID: 24781528 DOI: 10.1039/c4dt00421c] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
Abstract
The survival of all urease and/or hydrogenase containing pathogens depends on the proper homeostasis of nickel. In the scope of this perspectives paper, details of Ni(2+) metabolism of Helicobacter pylori, a widespread stomach-ulcer causing bacterium, are described. Nickel binding proteins and thermodynamics of such metal complexes are discussed in detail and special focus is given to potential nickel binding sequences in this metal's chaperones and regulators. A list of potential Ni(2+) binding sites in various pathogens is presented, which points out numerous examples of nickel interactions that still need to be understood.
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17
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Merloni A, Dobrovolska O, Zambelli B, Agostini F, Bazzani M, Musiani F, Ciurli S. Molecular landscape of the interaction between the urease accessory proteins UreE and UreG. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2014; 1844:1662-74. [PMID: 24982029 DOI: 10.1016/j.bbapap.2014.06.016] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/23/2014] [Revised: 06/12/2014] [Accepted: 06/19/2014] [Indexed: 01/07/2023]
Abstract
Urease, the most efficient enzyme so far discovered, depends on the presence of nickel ions in the catalytic site for its activity. The transformation of inactive apo-urease into active holo-urease requires the insertion of two Ni(II) ions in the substrate binding site, a process that involves the interaction of four accessory proteins named UreD, UreF, UreG and UreE. This study, carried out using calorimetric and NMR-based structural analysis, is focused on the interaction between UreE and UreG from Sporosarcina pasteurii, a highly ureolytic bacterium. Isothermal calorimetric protein-protein titrations revealed the occurrence of a binding event between SpUreE and SpUreG, entailing two independent steps with positive cooperativity (Kd1=42±9μM; Kd2=1.7±0.3μM). This was interpreted as indicating the formation of the (UreE)2(UreG)2 hetero-oligomer upon binding of two UreG monomers onto the pre-formed UreE dimer. The molecular details of this interaction were elucidated using high-resolution NMR spectroscopy. The occurrence of SpUreE chemical shift perturbations upon addition of SpUreG was investigated and analyzed to establish the protein-protein interaction site. The latter appears to involve the Ni(II) binding site as well as mobile portions on the C-terminal and the N-terminal domains. Docking calculations based on the information obtained from NMR provided a structural basis for the protein-protein contact site. The high sequence and structural similarity within these protein classes suggests a generality of the interaction mode among homologous proteins. The implications of these results on the molecular details of the urease activation process are considered and analyzed.
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Affiliation(s)
- Anna Merloni
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Italy
| | - Olena Dobrovolska
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Italy
| | - Barbara Zambelli
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Italy
| | - Federico Agostini
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Italy
| | - Micaela Bazzani
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Italy
| | - Francesco Musiani
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Italy
| | - Stefano Ciurli
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Italy.
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18
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Abstract
The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4 (+). This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg(2+) at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium.
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19
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Twin-arginine translocation system in Helicobacter pylori: TatC, but not TatB, is essential for viability. mBio 2014; 5:e01016-13. [PMID: 24449753 PMCID: PMC3903283 DOI: 10.1128/mbio.01016-13] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
The twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects. This work reports the first characterization of the twin-arginine translocation (Tat) system in the gastric pathogen Helicobacter pylori. While tatB mutants were easily obtained, only single-crossover partial tatC mutants or conditional tatC mutants could be generated, indicating that tatC is essential in H. pylori, a surprising finding given the fact that only four proteins are predicted to be translocated by the Tat system in this bacterium. The levels of activity of hydrogenase and catalase, two of the predicted Tat-dependent enzymes, were affected in these mutants. In addition, all tat mutants displayed cell envelope defects, and tatC mutants were deficient in mouse colonization.
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20
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H. pylori virulence factors: influence on immune system and pathology. Mediators Inflamm 2014; 2014:426309. [PMID: 24587595 PMCID: PMC3918698 DOI: 10.1155/2014/426309] [Citation(s) in RCA: 74] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2013] [Accepted: 12/19/2013] [Indexed: 02/07/2023] Open
Abstract
Helicobacter pylori is the most widespread chronic bacterial agent in humans and is well recognized for its association with ulcer disease and gastric cancer, with both representing major global health and socioeconomic issues. Given the high level of adaptation and the coevolution of this bacterium with its human host, a thorough and multidirectional view of the specific microbiological characteristics of this infection as well as the host physiology is needed in order to develop novel means of prevention of therapy. This review aims to pinpoint some of these potentially important angles, which have to be considered mutually when studying H. pylori's pathogenicity. The host's biological changes due to the virulence factors are a valuable pillar of H. pylori research as are the mechanisms by which bacteria provoke these changes. In this context, necessary adhesion molecules and significant virulence factors of H. pylori are discussed. Moreover, metabolism of the bacteria, one of the most important aspects for a better understanding of bacterial physiology and consequently possible therapeutic and prophylactic strategies, is addressed. On the other hand, we discuss the recent experimental proofs of the "hygiene hypothesis" in correlation with Helicobacter's infection, which adds another aspect of complexity to this infection.
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21
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Selectivity of Ni(II) and Zn(II) binding to Sporosarcina pasteurii UreE, a metallochaperone in the urease assembly: a calorimetric and crystallographic study. J Biol Inorg Chem 2013; 18:1005-17. [PMID: 24126709 DOI: 10.1007/s00775-013-1049-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2013] [Accepted: 09/13/2013] [Indexed: 10/26/2022]
Abstract
Urease is a nickel-dependent enzyme that plays a critical role in the biogeochemical nitrogen cycle by catalyzing the hydrolysis of urea to ammonia and carbamate. This enzyme, initially synthesized in the apo form, needs to be activated by incorporation of two nickel ions into the active site, a process driven by the dimeric metallochaperone UreE. Previous studies reported that this protein can bind different metal ions in vitro, beside the cognate Ni(II). This study explores the metal selectivity and affinity of UreE from Sporosarcina pasteurii (Sp, formerly known as Bacillus pasteurii) for cognate [Ni(II)] and noncognate [Zn(II)] metal ions. In particular, the thermodynamic parameters of SpUreE Ni(II) and Zn(II) binding have been determined using isothermal titration calorimetry. These experiments show that two Ni(II) ions bind to the protein dimer with positive cooperativity. The high-affinity site involves the conserved solvent-exposed His(100) and the C-terminal His(145), whereas the low-affinity site comprises also the C-terminal His(147). Zn(II) binding to the protein, occurring in the same protein regions and with similar affinity as compared to Ni(II), causes metal-driven dimerization of the protein dimer. The crystal structure of the protein obtained in the presence of equimolar amounts of both metal ions indicates that the high-affinity metal binding site binds Ni(II) preferentially over Zn(II). The ability of the protein to select Ni(II) over Zn(II) was confirmed by competition experiments in solution as well as by analysis of X-ray anomalous dispersion data. Overall, the thermodynamics and structural parameters that modulate the metal ion specificity of the different binding sites on the protein surface of SpUreE have been established.
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22
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Structure of UreG/UreF/UreH complex reveals how urease accessory proteins facilitate maturation of Helicobacter pylori urease. PLoS Biol 2013; 11:e1001678. [PMID: 24115911 PMCID: PMC3792862 DOI: 10.1371/journal.pbio.1001678] [Citation(s) in RCA: 81] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2013] [Accepted: 08/29/2013] [Indexed: 11/19/2022] Open
Abstract
Structural and biochemical study of urease accessory protein complex provides mechanistic insights into the delivery of nickel to metalloenzyme urease, an enzyme enabling the survival of Helicobacter pylori in the human stomach. Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease. Catalytic activities of many important enzymes depend upon metal cofactors. Ensuring each enzyme acquires the proper type of metal cofactor is essential to life. One such example is urease, which is a nickel containing metalloenzyme catalyzing the hydrolysis of urea to ammonia. The survival of Helicobacter pylori, a stomach ulcer–causing pathogen, in the human stomach depends on the ammonia released to neutralize gastric acid. In this study, we revealed the detail mechanism of how urease accessory proteins UreF, UreH, and UreG cooperate to couple GTP hydrolysis to deliver nickel to urease. UreF/UreH complex interacts with two molecules of GTPase UreG and assembles a metal binding site located at the interface between two UreG molecules. Nickel can induce GTP-dependent dimerization of UreG. This nickel-carrying UreG dimer together with UreF, UreH, and urease assemble into a protein complex. Upon stimulation of UreG GTPase activity by bicarbonate, UreG hydrolyses GTP and releases nickel into urease. Other nickel-delivering NTPases share similar properties with UreG; therefore, the nickel delivery mechanism described here is likely universally shared among these proteins.
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23
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Cheng T, Xia W, Wang P, Huang F, Wang J, Sun H. Histidine-rich proteins in prokaryotes: metal homeostasis and environmental habitat-related occurrence. Metallomics 2013; 5:1423-1429. [PMID: 23925314 DOI: 10.1039/c3mt00059a] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
Abstract
Increasing amounts of histidine-rich proteins (HRPs) have been found in microorganisms. We systematically analyzed the proteomes of 675 prokaryotes including 52 archaea and 623 bacteria for histidine-rich motifs (HRMs). We show that HRPs are widespread in prokaryotic proteomes, with the majority being involved in metal homeostasis. HRPs are frequently found in the proteomes of certain orders of rhizobia and pathogenic Gram-negative bacteria, but are essentially absent in obligate intracellular pathogenic species. The occurrence of HRPs in the proteomes of prokaryotes is related to their habitats. We further revealed a class of globally histidine-rich bacterial proteins. This approach can readily be used to identify other single amino acid rich motifs (and proteins) in microbial proteomes to facilitate the exploration of their functions.
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Affiliation(s)
- Tianfan Cheng
- Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P.R. China.
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24
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25
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Rowinska-Zyrek M, Witkowska D, Potocki S, Remelli M, Kozlowski H. His-rich sequences – is plagiarism from nature a good idea? NEW J CHEM 2013. [DOI: 10.1039/c2nj40558j] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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26
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Higgins KA, Carr CE, Maroney MJ. Specific metal recognition in nickel trafficking. Biochemistry 2012; 51:7816-32. [PMID: 22970729 DOI: 10.1021/bi300981m] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Nickel is an essential metal for a number of bacterial species that have developed systems for acquiring, delivering, and incorporating the metal into target enzymes and controlling the levels of nickel in cells to prevent toxic effects. As with other transition metals, these trafficking systems must be able to distinguish between the desired metal and other transition metal ions with similar physical and chemical properties. Because there are few enzymes (targets) that require nickel for activity (e.g., Escherichia coli transports nickel for hydrogenases made under anaerobic conditions, and Helicobacter pylori requires nickel for hydrogenase and urease that are essential for acid viability), the "traffic pattern" for nickel is relatively simple, and nickel trafficking therefore presents an opportunity to examine a system for the mechanisms that are used to distinguish nickel from other metals. In this review, we describe the details known for examples of uptake permeases, metallochaperones and proteins involved in metallocenter assembly, and nickel metalloregulators. We also illustrate a variety of mechanisms, including molecular recognition in the case of NikA protein and examples of allosteric regulation for HypA, NikR, and RcnR, employed to generate specific biological responses to nickel ions.
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Affiliation(s)
- Khadine A Higgins
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
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27
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Benoit SL, McMurry JL, Hill SA, Maier RJ. Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition. Biochim Biophys Acta Gen Subj 2012; 1820:1519-25. [PMID: 22698670 DOI: 10.1016/j.bbagen.2012.06.002] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2012] [Revised: 06/01/2012] [Accepted: 06/04/2012] [Indexed: 01/15/2023]
Abstract
BACKGROUND The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni(2+) into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH. METHODS Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG. RESULTS Using both methods, affinity constants were found to be 5nM and 13nM for HypA-UreE and 8μM and 14μM for UreG-UreE. Neither Zn(2+) nor Ni(2+) had an effect on the kinetics or stability of the HypA-UreE complex. By contrast, addition of Zn(2+), but not Ni(2+), altered the kinetics and greatly increased the stability of the UreE-UreG complex, likely due in part to Zn(2+)-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition. GENERAL SIGNIFICANCE Factors influencing the pathogen's nickel budget are important to understand pathogenesis and for future drug design.
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Affiliation(s)
- Stéphane L Benoit
- Department of Microbiology, University of Georgia, 805 Biological Sciences Bldg., Athens, GA, USA
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Crystallographic and X-ray absorption spectroscopic characterization of Helicobacter pylori UreE bound to Ni²⁺ and Zn²⁺ reveals a role for the disordered C-terminal arm in metal trafficking. Biochem J 2012; 441:1017-26. [PMID: 22010876 DOI: 10.1042/bj20111659] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni²⁺ ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni²⁺ insertion into the apoenzyme. Crystals of apo-HpUreE (H. pylori UreE) and its Ni⁺- and Zn⁺-bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 Å (apo), 1.59 Å (Ni²⁺) and 2.52 Å (Zn²⁺) resolution, show the conserved proximal and solvent-exposed His¹⁰² residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apoprotein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His¹⁵². The analysis of X-ray absorption spectral data obtained using solutions of Ni²⁺- and Zn²⁺-bound HpUreE provided accurate information of the metal-ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal-ion binding, and the mutual influence of protein framework and metal-ion stereo-electronic properties in establishing co-ordination number and geometry leading to metal selectivity.
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Martin-Diaconescu V, Bellucci M, Musiani F, Ciurli S, Maroney MJ. Unraveling the Helicobacter pylori UreG zinc binding site using X-ray absorption spectroscopy (XAS) and structural modeling. J Biol Inorg Chem 2011; 17:353-61. [PMID: 22068961 DOI: 10.1007/s00775-011-0857-9] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2011] [Accepted: 10/24/2011] [Indexed: 01/22/2023]
Abstract
The pathogenicity of Helicobacter pylori depends on the activity of urease for pH modification. Urease activity requires assembly of a dinickel active site that is facilitated in part by GTP hydrolysis by UreG. The proper functioning of Helicobacter pylori UreG (HpUreG) is dependent on Zn(II) binding and dimerization. X-ray absorption spectroscopy and structural modeling were used to elucidate the structure of the Zn(II) site in HpUreG. These studies independently indicated a site at the dimer interface that has trigonal bipyramidal geometry and is composed of two axial cysteines at 2.29(2) Å, two equatorial histidines at 1.99(1) Å, and a solvent-accessible coordination site. The final model for the Zn(II) site structure was determined by refining multiple-scattering extended X-ray absorption fine structure fits using the geometry predicted by homology modeling and ab initio calculations.
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30
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Cheng T, Li H, Xia W, Sun H. Multifaceted SlyD from Helicobacter pylori: implication in [NiFe] hydrogenase maturation. J Biol Inorg Chem 2011; 17:331-43. [PMID: 22045417 PMCID: PMC3292732 DOI: 10.1007/s00775-011-0855-y] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2011] [Accepted: 10/07/2011] [Indexed: 12/31/2022]
Abstract
SlyD belongs to the FK506-binding protein (FKBP) family with both peptidylprolyl isomerase (PPIase) and chaperone activities, and is considered to be a ubiquitous cytosolic protein-folding facilitator in bacteria. It possesses a histidine- and cysteine-rich C-terminus binding to selected divalent metal ions (e.g., Ni2+, Zn2+), which is important for its involvement in the maturation processes of metalloenzymes. We have determined the solution structure of C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC). HpSlyDΔC folds into two well-separated, orientation-independent domains: the PPIase-active FKBP domain and the chaperone-active insert-in-flap (IF) domain. The FKBP domain consists of a four-stranded antiparallel β-sheet with an α-helix on one side, whereas the IF domain folds into a four-stranded antiparallel β-sheet accompanied by a short α-helix. Intact H. pylori SlyD binds both Ni2+ and Zn2+, with dissociation constants of 2.74 and 3.79 μM respectively. Intriguingly, binding of Ni2+ instead of Zn2+ induces protein conformational changes around the active sites of the FKBP domain, implicating a regulatory role of nickel. The twin-arginine translocation (Tat) signal peptide from the small subunit of [NiFe] hydrogenase (HydA) binds the protein at the IF domain. Nickel binding and the recognition of the Tat signal peptide by the protein suggest that SlyD participates in [NiFe] hydrogenase maturation processes.
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Affiliation(s)
- Tianfan Cheng
- Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China
| | - Hongyan Li
- Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China
| | - Wei Xia
- Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China
| | - Hongzhe Sun
- Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China
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31
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Ge R, Sun X. The in vivo functions of a histidine-rich protein Hpn in Helicobacter pylori: linking gastric and Alzheimer's diseases together? Med Hypotheses 2011; 77:788-90. [PMID: 21852052 DOI: 10.1016/j.mehy.2011.07.038] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2011] [Accepted: 07/18/2011] [Indexed: 12/17/2022]
Abstract
Helicobacter pylori causes such gastric diseases as gastritis, peptic ulcerations, gastric cancer and MALT lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms amyloid-like oligomers in physiologically relevant conditions. Here we proposed the in vivo functions of this protein with relevance to its physical locations. The collective evidence presented here shed some light on the pathologic mechanisms of H. pylori infections, with emphasis on the bacterial colonization in the gastric environment, pathological effects to the gastric epithelial cells and the possible link to Alzheimer's disease.
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Affiliation(s)
- Ruiguang Ge
- The Laboratory of Integrative Biosciences, College of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China.
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32
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Zambelli B, Musiani F, Benini S, Ciurli S. Chemistry of Ni2+ in urease: sensing, trafficking, and catalysis. Acc Chem Res 2011; 44:520-30. [PMID: 21542631 DOI: 10.1021/ar200041k] [Citation(s) in RCA: 181] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Transition metals are both essential to enzymatic catalysis and limited in environmental availability. These two biological facts have together driven organisms to evolve mechanisms for selective metal ion sensing and utilization. Changes in metal ion concentrations are perceived by metal-dependent transcription factors and transduced into appropriate cellular responses, which regulate the machineries of competitive metal ion homeostasis and metallo-enzyme activation. The intrinsic toxicity of the majority of metal ions further creates a need for regulated intracellular trafficking, which is carried out by specific chaperones. The Ni(2+)-dependent urease enzymatic system serves as a paradigm for studying the strategies that cells use to handle an essential, yet toxic, metal ion. Although the discovery of urease as the first biological system for which nickel is essential for activity dates to 1975, the rationale for Ni(2+) selection, as well as the cascade of events involving metal-dependent gene regulation and protein-protein interactions leading to enzyme activation, have yet to be fully unraveled. The past 14 years since the Account by Hausinger and co-workers (Karplus, P. A.; Pearson, M. A.; Hausinger, R. P. Acc. Chem. Res. 1997, 30, 330-337) have witnessed impressive achievements in the understanding of the biological chemistry of Ni(2+) in the urease system. In our Account, we discuss more recent advances in the comprehension of the specific role of Ni(2+) in the catalysis and the interplay between Ni(2+) and other metal ions, such as Zn(2+) and Fe(2+), in the metal-dependent enzyme activity. Our discussion focuses on work carried out in our laboratory. In particular, the structural features of the enzyme bound to inhibitors, substrate analogues, and transition state or intermediate analogues have shed light on the catalytic mechanism. Structural and functional information has been correlated to understand the Ni(2+) sensing effected by NikR, a nickel-dependent transcription factor. The urease activation process, involving insertion of Ni(2+) into the urease active site, has been in part dissected and analyzed through the investigation of the molecular properties of the accessory proteins UreD, UreF, and UreG. The intracellular trafficking of Ni(2+) has been rationalized through a deeper understanding of the structural and metal-binding properties of the metallo-chaperone UreE. All the while, a number of key general concepts have been revealed and developed. These include an understanding of (i) the overall ancillary role of Zn(2+) in nickel metabolism, (ii) the intrinsically disordered nature of the GTPase responsible for coupling the energy consumption to the carbon dioxide requirement for the urease activation process, and (iii) the role of the accessory proteins regulating this GTPase activity.
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Affiliation(s)
- Barbara Zambelli
- Laboratory of Bioinorganic Chemistry, University of Bologna, Italy
| | | | - Stefano Benini
- Faculty of Science and Technology, Free University of Bolzano, Italy
| | - Stefano Ciurli
- Laboratory of Bioinorganic Chemistry, University of Bologna, Italy
- CERM (Center of Magnetic Resonance), University of Florence, Italy
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Carter EL, Flugga N, Boer JL, Mulrooney SB, Hausinger RP. Interplay of metal ions and urease. Metallomics 2011; 1:207-21. [PMID: 20046957 DOI: 10.1039/b903311d] [Citation(s) in RCA: 127] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Urease, the first enzyme to be crystallized, contains a dinuclear nickel metallocenter that catalyzes the decomposition of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel versus other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel metallochaperones and GTP-dependent molecular chaperones or play other roles in the maturation process. In addition, some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.
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Affiliation(s)
- Eric L Carter
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-4320, USA
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34
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Lam R, Romanov V, Johns K, Battaile KP, Wu-Brown J, Guthrie JL, Hausinger RP, Pai EF, Chirgadze NY. Crystal structure of a truncated urease accessory protein UreF from Helicobacter pylori. Proteins 2011; 78:2839-48. [PMID: 20635345 DOI: 10.1002/prot.22802] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Urease plays a central role in the pathogenesis of Helicobacter pylori in humans. Maturation of this nickel metalloenzyme in bacteria requires the participation of the accessory proteins UreD (termed UreH in H. pylori), UreF, and UreG, which form sequential complexes with the urease apoprotein as well as UreE, a metallochaperone. Here, we describe the crystal structure of C-terminal truncated UreF from H. pylori (residues 1-233), the first UreF structure to be determined, at 1.55 A resolution using SAD methods. UreF forms a dimer in vitro and adopts an all-helical fold congruent with secondary structure prediction. On the basis of evolutionary conservation analysis, the structure reveals a probable binding surface for interaction with other urease components as well as key conserved residues of potential functional relevance.
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Affiliation(s)
- Robert Lam
- Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada
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Sydor AM, Liu J, Zamble DB. Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease. J Bacteriol 2011; 193:1359-68. [PMID: 21239585 PMCID: PMC3067625 DOI: 10.1128/jb.01333-10] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2010] [Accepted: 01/08/2011] [Indexed: 01/12/2023] Open
Abstract
The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.
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Affiliation(s)
- Andrew M. Sydor
- Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada
| | - Jenny Liu
- Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada
| | - Deborah B. Zamble
- Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada
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36
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Ahmad MSA, Ashraf M. Essential roles and hazardous effects of nickel in plants. REVIEWS OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY 2011; 214:125-167. [PMID: 21913127 DOI: 10.1007/978-1-4614-0668-6_6] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
With the world's ever increasing human population, the issues related to environmental degradation of toxicant chemicals are becoming more serious. Humans have accelerated the emission to the environment of many organic and inorganic pollutants such as pesticides, salts, petroleum products, acids, heavy metals, etc. Among different environmental heavy-metal pollutants, Ni has gained considerable attention in recent years, because of its rapidly increasing concentrations in soil, air, and water in different parts of the world. The main mechanisms by which Ni is taken up by plants are passive diffusion and active transport. Soluble Ni compounds are preferably absorbed by plants passively, through a cation transport system; chelated Ni compounds are taken up through secondary, active-transport-mediated means, using transport proteins such as permeases. Insoluble Ni compounds primarily enter plant root cells through endocytosis. Once absorbed by roots, Ni is easily transported to shoots via the xylem through the transpiration stream and can accumulate in neonatal parts such as buds, fruits, and seeds. The Ni transport and retranslocation processes are strongly regulated by metal-ligand complexes (such as nicotianamine, histidine, and organic acids) and by some proteins that specifically bind and transport Ni. Nickel, in low concentrations, fulfills a variety of essential roles in plants, bacteria, and fungi. Therefore, Ni deficiency produces an array of effects on growth and metabolism of plants, including reduced growth, and induction of senescence, leaf and meristem chlorosis, alterations in N metabolism, and reduced Fe uptake. In addition, Ni is a constituent of several metallo-enzymes such as urease, superoxide dismutase, NiFe hydrogenases, methyl coenzyme M reductase, carbon monoxide dehydrogenase, acetyl coenzyme-A synthase, hydrogenases, and RNase-A. Therefore, Ni deficiencies in plants reduce urease activity, disturb N assimilation, and reduce scavenging of superoxide free radical. In bacteria, Ni participates in several important metabolic reactions such as hydrogen metabolism, methane biogenesis, and acetogenesis. Although Ni is metabolically important in plants, it is toxic to most plant species when present at excessive amounts in soil and in nutrient solution. High Ni concentrations in growth media severely retards seed germinability of many crops. This effect of Ni is a direct one on the activities of amylases, proteases, and ribonucleases, thereby affecting the digestion and mobilization of food reserves in germinating seeds. At vegetative stages, high Ni concentrations retard shoot and root growth, affect branching development, deform various plant parts, produce abnormal flower shape, decrease biomass production, induce leaf spotting, disturb mitotic root tips, and produce Fe deficiency that leads to chlorosis and foliar necrosis. Additionally, excess Ni also affects nutrient absorption by roots, impairs plant metabolism, inhibits photosynthesis and transpiration, and causes ultrastructural modifications. Ultimately, all of these altered processes produce reduced yields of agricultural crops when such crops encounter excessive Ni exposures.
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Herbst RW, Perovic I, Martin-Diaconescu V, O'Brien K, Chivers PT, Pochapsky SS, Pochapsky TC, Maroney MJ. Communication between the zinc and nickel sites in dimeric HypA: metal recognition and pH sensing. J Am Chem Soc 2010; 132:10338-51. [PMID: 20662514 DOI: 10.1021/ja1005724] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
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)(4) site to a Zn(His)(2)(Cys)(2) site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the beta-CH(2) 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)(4)) 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.
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Affiliation(s)
- Robert W Herbst
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
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38
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Shi R, Munger C, Asinas A, Benoit SL, Miller E, Matte A, Maier RJ, Cygler M. Crystal structures of apo and metal-bound forms of the UreE protein from Helicobacter pylori: role of multiple metal binding sites. Biochemistry 2010; 49:7080-8. [PMID: 20681615 DOI: 10.1021/bi100372h] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The crystal structure of the urease maturation protein UreE from Helicobacter pylori has been determined in its apo form at 2.1 A resolution, bound to Cu(2+) at 2.7 A resolution, and bound to Ni(2+) at 3.1 A resolution. Apo UreE forms dimers, while the metal-bound enzymes are arranged as tetramers that consist of a dimer of dimers associated around the metal ion through coordination by His102 residues from each subunit of the tetramer. Comparison of independent subunits from different crystal forms indicates changes in the relative arrangement of the N- and C-terminal domains in response to metal binding. The improved ability of engineered versions of UreE containing hexahistidine sequences at either the N-terminal or C-terminal end to provide Ni(2+) for the final metal sink (urease) is eliminated in the H102A version. Therefore, the ability of the improved Ni(2+)-binding versions to deliver more nickel is likely an effect of an increased local concentration of metal ions that can rapidly replenish transferred ions bound to His102.
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Affiliation(s)
- Rong Shi
- Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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Characterization of the Klebsiella aerogenes urease accessory protein UreD in fusion with the maltose binding protein. J Bacteriol 2010; 192:2294-304. [PMID: 20207756 DOI: 10.1128/jb.01426-09] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a DeltaureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (> 670 kDa) that bound approximately 2.5 Ni2+ ions (K(d) of approximately 50 microM, where K(d) is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (approximately 4 Zn2+ ions per protomer; K(d) of 5 microM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.
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Schauer K, Muller C, Carrière M, Labigne A, Cavazza C, De Reuse H. The Helicobacter pylori GroES cochaperonin HspA functions as a specialized nickel chaperone and sequestration protein through its unique C-terminal extension. J Bacteriol 2010; 192:1231-7. [PMID: 20061471 PMCID: PMC2820833 DOI: 10.1128/jb.01216-09] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2009] [Accepted: 12/27/2009] [Indexed: 02/06/2023] Open
Abstract
The transition metal nickel plays a central role in the human gastric pathogen Helicobacter pylori because it is required for two enzymes indispensable for colonization, the nickel metalloenzyme urease and [NiFe] hydrogenase. To sustain nickel availability for these metalloenzymes while providing protection from the metal's harmful effects, H. pylori is equipped with several specific nickel-binding proteins. Among these, H. pylori possesses a particular chaperone, HspA, that is a homolog of the highly conserved and essential bacterial heat shock protein GroES. HspA contains a unique His-rich C-terminal extension and was demonstrated to bind nickel in vitro. To investigate the function of this extension in H. pylori, we constructed mutants carrying either a complete deletion or point mutations in critical residues of this domain. All mutants presented a decreased intracellular nickel content measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced nickel tolerance. While urease activity was unaffected in the mutants, [NiFe] hydrogenase activity was significantly diminished when the C-terminal extension of HspA was mutated. We conclude that H. pylori HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in nickel-dependent maturation of hydrogenase.
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Affiliation(s)
- Kristine Schauer
- Institut Pasteur, Département de Microbiologie, Unité Postulante Pathogenèse de Helicobacter, 75724 Paris Cedex 15, France, Institut Pasteur, Département de Microbiologie, Unité de Pathogénie Bactérienne des Muqueuses, 75724 Paris Cedex 15, France, UMR3299 CEA-CNRS SIS2M, LSDRNI CEA/Saclay, 91191 Gif-sur-Yvette, France, Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France
| | - Cécile Muller
- Institut Pasteur, Département de Microbiologie, Unité Postulante Pathogenèse de Helicobacter, 75724 Paris Cedex 15, France, Institut Pasteur, Département de Microbiologie, Unité de Pathogénie Bactérienne des Muqueuses, 75724 Paris Cedex 15, France, UMR3299 CEA-CNRS SIS2M, LSDRNI CEA/Saclay, 91191 Gif-sur-Yvette, France, Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France
| | - Marie Carrière
- Institut Pasteur, Département de Microbiologie, Unité Postulante Pathogenèse de Helicobacter, 75724 Paris Cedex 15, France, Institut Pasteur, Département de Microbiologie, Unité de Pathogénie Bactérienne des Muqueuses, 75724 Paris Cedex 15, France, UMR3299 CEA-CNRS SIS2M, LSDRNI CEA/Saclay, 91191 Gif-sur-Yvette, France, Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France
| | - Agnès Labigne
- Institut Pasteur, Département de Microbiologie, Unité Postulante Pathogenèse de Helicobacter, 75724 Paris Cedex 15, France, Institut Pasteur, Département de Microbiologie, Unité de Pathogénie Bactérienne des Muqueuses, 75724 Paris Cedex 15, France, UMR3299 CEA-CNRS SIS2M, LSDRNI CEA/Saclay, 91191 Gif-sur-Yvette, France, Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France
| | - Christine Cavazza
- Institut Pasteur, Département de Microbiologie, Unité Postulante Pathogenèse de Helicobacter, 75724 Paris Cedex 15, France, Institut Pasteur, Département de Microbiologie, Unité de Pathogénie Bactérienne des Muqueuses, 75724 Paris Cedex 15, France, UMR3299 CEA-CNRS SIS2M, LSDRNI CEA/Saclay, 91191 Gif-sur-Yvette, France, Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France
| | - Hilde De Reuse
- Institut Pasteur, Département de Microbiologie, Unité Postulante Pathogenèse de Helicobacter, 75724 Paris Cedex 15, France, Institut Pasteur, Département de Microbiologie, Unité de Pathogénie Bactérienne des Muqueuses, 75724 Paris Cedex 15, France, UMR3299 CEA-CNRS SIS2M, LSDRNI CEA/Saclay, 91191 Gif-sur-Yvette, France, Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France
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Duckworth MJ, Okoli AS, Mendz GL. Novel Helicobacter pylori therapeutic targets: the unusual suspects. Expert Rev Anti Infect Ther 2009; 7:835-67. [PMID: 19735225 DOI: 10.1586/eri.09.61] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Understanding the current status of the discovery and development of anti-Helicobacter therapies requires an overview of the searches for therapeutic targets performed to date. A summary is given of the very substantial body of work conducted in the quest to find Helicobacter pylori genes that could be suitable candidates for therapeutic intervention. The products of most of these genes perform metabolic functions, and others have roles in growth, cell motility and colonization. The genes identified as potential targets have been organized into three categories according to their degree of characterization. A short description and evaluation is provided of the main candidates in each category. Investigations of potential therapeutic targets have generated a wealth of information about the physiology and genetics of H. pylori, and its interactions with the host, but have yielded little by way of new therapies.
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Affiliation(s)
- Megan J Duckworth
- School of Medicine, Sydney, The University of Notre Dame Australia, 160 Oxford Street, Darlinghurst, NSW 2010, Australia.
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Haas CE, Rodionov DA, Kropat J, Malasarn D, Merchant SS, de Crécy-Lagard V. A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life. BMC Genomics 2009; 10:470. [PMID: 19822009 PMCID: PMC2770081 DOI: 10.1186/1471-2164-10-470] [Citation(s) in RCA: 121] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2009] [Accepted: 10/12/2009] [Indexed: 11/11/2022] Open
Abstract
Background COG0523 proteins are, like the nickel chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523-encoding gene, cobW, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family. Results Based on a combination of comparative genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at least fifteen subgroups. The CobW subgroup involved in cobalamin synthesis represents only one small sub-fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to zinc limitation based on the presence of the corresponding COG0523-encoding genes downstream from putative Zur binding sites in many bacterial genomes. Zur binding sites in these genomes are also associated with candidate zinc-independent paralogs of zinc-dependent enzymes. Finally, the potential role of COG0523 in zinc homeostasis is not limited to Bacteria. We have predicted a link between COG0523 and regulation by zinc in Archaea and show that two COG0523 genes are induced upon zinc depletion in a eukaryotic reference organism, Chlamydomonas reinhardtii. Conclusion This work lays the foundation for the pursuit by experimental methods of the specific role of COG0523 members in metal trafficking. Based on phylogeny and comparative genomics, both the metal specificity and the protein target(s) might vary from one COG0523 subgroup to another. Additionally, Zur-dependent expression of COG0523 and putative paralogs of zinc-dependent proteins may represent a mechanism for hierarchal zinc distribution and zinc sparing in the face of inadequate zinc nutrition.
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Affiliation(s)
- Crysten E Haas
- Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA.
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Affiliation(s)
- Yanjie Li
- Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada
| | - Deborah B. Zamble
- Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada
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Helicobacter pylori UreE, a urease accessory protein: specific Ni(2+)- and Zn(2+)-binding properties and interaction with its cognate UreG. Biochem J 2009; 422:91-100. [PMID: 19476442 DOI: 10.1042/bj20090434] [Citation(s) in RCA: 73] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni(2+) for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni(2+) insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni(2+) (Kd=0.15 microM) or Zn(2+) (Kd=0.49 microM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn(2+) and Ni(2+), which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE-HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn(2+), and not for Ni(2+), in the stabilization of this complex was demonstrated using size-exclusion chromatography, light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of a novel binding site for Zn(2+), actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn(2+) in the Ni(2+)-dependent urease system is envisaged.
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An intact urease assembly pathway is required to compete with NikR for nickel ions in Helicobacter pylori. J Bacteriol 2009; 191:2405-8. [PMID: 19168618 DOI: 10.1128/jb.01657-08] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We examined the effects of urease and hydrogenase assembly gene deletions on NikR activation in H. pylori strains 26695 and G27. The loss of any component of urease assembly increased NikR activity under Ni2+-limiting conditions, as measured by reduced transcript levels and 63Ni accumulation. Additionally, SlyD functioned in urease assembly in strain 26695.
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Jones KR, Cha JH, Merrell DS. Who's Winning the War? Molecular Mechanisms of Antibiotic Resistance in Helicobacter pylori. CURRENT DRUG THERAPY 2008; 3:190-203. [PMID: 21765819 DOI: 10.2174/157488508785747899] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The ability of clinicians to wage an effective war against many bacterial infections is increasingly being hampered by skyrocketing rates of antibiotic resistance. Indeed, antibiotic resistance is a significant problem for treatment of diseases caused by virtually all known infectious bacteria. The gastric pathogen Helicobacter pylori is no exception to this rule. With more than 50% of the world's population infected, H. pylori exacts a tremendous medical burden and represents an interesting paradigm for cancer development; it is the only bacterium that is currently recognized as a carcinogen. It is now firmly established that H. pylori infection is associated with diseases such as gastritis, peptic and duodenal ulceration and two forms of gastric cancer, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. With such a large percentage of the population infected, increasing rates of antibiotic resistance are particularly vexing for a treatment regime that is already fairly complicated; treatment consists of two antibiotics and a proton pump inhibitor. To date, resistance has been found to all primary and secondary lines of antibiotic treatment as well as to drugs used for rescue therapy.
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Affiliation(s)
- Kathleen R Jones
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA
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Paksi Z, Jancsó A, Pacello F, Nagy N, Battistoni A, Gajda T. Copper and zinc binding properties of the N-terminal histidine-rich sequence of Haemophilus ducreyi Cu,Zn superoxide dismutase. J Inorg Biochem 2008; 102:1700-10. [PMID: 18565588 DOI: 10.1016/j.jinorgbio.2008.04.007] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2008] [Revised: 04/23/2008] [Accepted: 04/25/2008] [Indexed: 10/22/2022]
Abstract
The Cu,Zn superoxide dismutase (Cu,ZnSOD) isolated from Haemophilus ducreyi possesses a His-rich N-terminal metal binding domain, which has been previously proposed to play a copper(II) chaperoning role. To analyze the metal binding ability and selectivity of the histidine-rich domain we have carried out thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first 11 amino acids of the enzyme (H(2)N-HGDHMHNHDTK-OH, L). This peptide has highly versatile metal binding ability and provides one and three high affinity binding sites for zinc(II) and copper(II), respectively. In equimolar solutions the MHL complexes are dominant in the neutral pH-range with protonated lysine epsilon-amino group. As a consequence of its multidentate nature, L binds zinc and copper with extraordinary high affinity (K(D,Zn)=1.6x10(-9)M and K(D,Cu)=5.0x10(-12)M at pH 7.4) and appears as the strongest zinc(II) and copper(II) chelator between the His-rich peptides so far investigated. These K(D) values support the already proposed role of the N-terminal His-rich region of H. ducreyi Cu,ZnSOD in copper recruitment under metal starvation, and indicate a similar function in the zinc(II) uptake, too. The kinetics of copper(II) transfer from L to the active site of Cu-free N-deleted H. ducreyi Cu,ZnSOD showed significant pH and copper-to-peptide ratio dependence, indicating specific structural requirements during the metal ion transfer to the active site. Interestingly, the complex CuHL has significant superoxide dismutase like activity, which may suggest multifunctional role of the copper(II)-bound N-terminal His-rich domain of H. ducreyi Cu,ZnSOD.
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Affiliation(s)
- Zoltán Paksi
- Department of Inorganic and Analytical Chemistry, University of Szeged, Szeged, Hungary
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Benoit SL, Zbell AL, Maier RJ. Nickel enzyme maturation in Helicobacter hepaticus: roles of accessory proteins in hydrogenase and urease activities. MICROBIOLOGY-SGM 2008; 153:3748-3756. [PMID: 17975083 DOI: 10.1099/mic.0.2007/010520-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, possesses a hydrogenase and a urease, both of which are nickel-containing enzymes. Analysis of the genome sequence of H. hepaticus revealed a full set of accessory genes which are required for the nickel maturation of each enzyme in other micro-organisms. Erythromycin-resistant mutants were constructed in four of these genes, hypA, hypB, ureE and ureG. Controls for polar effect were provided for hypA or hypB mutants by disrupting each gene located immediately downstream, i.e. hp0809 or hypC, respectively. Urease and hydrogenase activities were determined for each strain with or without supplemented nickel in the medium. As expected, the ureE and the ureG mutants had negligible urease activity, but they retained normal levels of hydrogenase activity. Urease levels could not be increased by the addition of nickel to the medium. The H. hepaticus hypA and hypB strains were deficient in both urease and hydrogenase activities, suggesting that both gene products act in a similar fashion as their counterparts in H. pylori. However, in contrast with the analogous mutants of H. pylori, the addition of nickel into the growth medium failed to restore either urease or hydrogenase enzyme levels in the H. hepaticus hypA or hypB mutants, indicating a probably unique role for these genes in the mouse liver pathogen.
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Affiliation(s)
| | - Andrea L Zbell
- Department of Microbiology, University of Georgia, Athens, GA, USA
| | - Robert J Maier
- Department of Microbiology, University of Georgia, Athens, GA, USA
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Ge R, Zhang Y, Sun X, Watt RM, He QY, Huang JD, Wilcox DE, Sun H. Thermodynamic and kinetic aspects of metal binding to the histidine-rich protein, Hpn. J Am Chem Soc 2007; 128:11330-1. [PMID: 16939237 DOI: 10.1021/ja062589t] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The histidine-rich protein, Hpn, binds to essential metals Ni2+, Cu2+, Zn2+ and a therapeutic metal Bi3+ with the in vitro affinities in the order of Cu2+ > Ni2+ > Bi3+ > Zn2+. In contrast, the in vivo (in E. coli) protection by the protein is in the order of Ni2+ > Bi3+ > Cu2+ approximately Zn2+. The release of Ni2+ from the protein follows a two-step process consisting of a rapidly established equilibrium and subsequently a rate-determining step (dissociation of Hpn-Ni...EDTA to Ni-EDTA). Our work suggests the nickel storage and homeostasis in H. pylori as the primary role of Hpn.
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Affiliation(s)
- Ruiguang Ge
- Department of Chemistry, University of Hong Kong, Pokfulam, Hong Kong, PRC
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