Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.4.6 (urease)
7,490 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To persist in the hostile acidic environment of the stomach, Helicobacter pylori must survive acid shock and grow at acidic pH. Of a library of 1250 random mutants screened for isolates unable to grow at low pH, 10 mutants were detected that were unable to grow at pH 4.8. However, all 10 mutants were resistant to acid shock. Four mutants had an insertion in genes of unknown function. One mutant was affected in lepA, an orthologue of a membrane GTPase. Three mutants were disrupted in loci involved in the transport of H(+) ions or other cations (FRaseI, czcA, and aldo-keto reductase). Two mutants were affected in loci that contribute to acid resistance in other microorganisms (uvrA and atpF'). Thus, at least 10 loci not related to urease are essential for the growth of H. pylori under acidic conditions and should be critical for lifelong infection by this pathogen.
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PMID:Identification of loci essential for the growth of Helicobacter pylori under acidic conditions. 1102 84

Previous studies demonstrated that two accessory proteins, HypA and HypB, play a role in nickel-dependent maturation of both hydrogenase and urease in Helicobacter pylori. Here, the two proteins were purified and characterized. HypA bound two Ni(2+) ions per dimer with positive cooperativity (Hill coefficient, approximately 2.0). The dissociation constants K(1) and K(2) for Ni(2+) were 58 and 1.3 microM, respectively. Studies on purified site-directed mutant proteins in each of the five histidine residues within HypA, revealed that only one histidine residue (His2) is vital for nickel binding. Nuclear magnetic resonance analysis showed that this purified mutant version (H2A) was similar in structure to that of the wild-type HypA protein. A chromosomal site-directed mutant of hypA (in the codon for His2) lacked hydrogenase activity and possessed only 2% of the wild-type urease activity. Purified HypB had a GTPase activity of 5 nmol of GTP hydrolyzed per nmol of HypB per min. Site-directed mutagenesis within the lysine residue in the conserved GTP-binding motif of HypB (Lys59) nearly abolished the GTPase activity of the mutant protein (K59A). In native solution, both HypA and HypB exist as homodimers with molecular masses of 25.8 and 52.4 kDa, respectively. However, a 1:1 molar mixture of HypA plus HypB gave rise to a 43.6-kDa species composed of both proteins. A 43-kDa heterodimeric HypA-HypB complex was also detected by cross-linking. The cross-linked adduct was still observed in the presence of 0.5 mM GTP or 1 microM nickel or when the mutant version of HypA (altered in His2) and HypB (altered in Lys59) were tested. Individually, HypA and HypB formed homodimeric cross-linked adducts. An interaction between HypA and the Hp0868 protein (encoded by the gene downstream of hypA) could not be detected via cross-linking, although such an interaction was predicted by yeast two-hybrid studies. In addition, the phenotype of an insertional mutation within the Hp0868 gene indicated that its presence is not critical for either the urease or the hydrogenase activity.
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PMID:Characterization of Helicobacter pylori nickel metabolism accessory proteins needed for maturation of both urease and hydrogenase. 1253 48

Nickel is a fundamental micronutrient for cellular life, but it is toxic in soluble form at nonphysiological concentrations. Such potentially contradictory features required living organisms to develop efficient systems for nickel utilization and homeostasis. This is the case for incorporation of nickel into the active site of urease, a multistep, tightly regulated process, requiring the interplay of various accessory proteins. The understanding of this activation mechanism may find medical applications against ureolytic bacteria, among which Mycobacterium tuberculosis is a deadly pathogen for humans. The topic of this study is UreG, an essential chaperone in the in vivo activation of urease upon insertion of Ni2+ into the active site. The protein was examined using both experimental and computational approaches. In particular, the soluble M. tuberculosis UreG (MtUreG) was overexpressed in Escherichia coli and purified to homogeneity. The identity of the isolated protein was established by mass spectrometry. On-line size-exclusion chromatography and light scattering indicated that MtUreG exists as a dimeric form in solution. Determination of the free thiol concentration revealed that a disulfide bond is present in the dimer. The isolated MtUreG shows low GTPase activity under native conditions, with a kcat of 0.01 min-1. Circular dichroism spectroscopy demonstrated the presence of a well-defined secondary structure (8% alpha-helices, 29% beta-strands) in MtUreG, whereas NMR spectroscopy indicated that this protein does not behave as a rigid three-dimensional fold and thus can be assigned to the class of intrinsically unstructured polypeptides. The molecular model of MtUreG in the fully folded and functional form was built using fold recognition algorithms. An extensive similarity search was performed to determine conservation patterns in all known bacterial UreG sequences. The generation of a multiple-sequence alignment and the related phylogenetic tree allowed us to recognize key residues and motifs that are likely important for protein function. A structural database containing the homology-built models of the most representative UreG proteins was created, confirming the structural analogies among the UreG family. A flexible region, likely to be important for protein function, is identified. The structural conservation among this class of GTPases is discussed on the basis of their function in the urease assembly process.
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PMID:Biochemical studies on Mycobacterium tuberculosis UreG and comparative modeling reveal structural and functional conservation among the bacterial UreG family. 1730 80

UreF is a protein that plays a role in the in vivo urease activation as a chaperone involved in the insertion of two Ni(2+) ions in the apo-urease active site. The molecular details of this process are unknown. In the absence of any molecular information on the UreF protein class, and as a step toward the comprehension of the relationships between UreF function and structure, we applied a structural modeling approach to infer useful biochemical knowledge on Bacillus pasteurii UreF (BpUreF). Similarity searches and multiple alignment of UreF protein sequences indicated that this class of proteins has a low homology with proteins of known structure. Fold recognition methods were therefore used to identify useful protein structural templates to model the structure of BpUreF. In particular, the templates belong to the class of GTPase-activating proteins. Modeling of BpUreF based on these templates was performed using the program MODELLER. The structure validation yielded good statistics, indicating that the model is plausible. This result suggests a role for UreF in urease active site biosynthesis as a regulator of the activity of UreG, a small G protein involved in the in vivo apo-urease activation process and established to catalyze GTP hydrolysis.
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PMID:A model-based proposal for the role of UreF as a GTPase-activating protein in the urease active site biosynthesis. 1751 Sep 59

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.
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PMID:Zn2+-linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation. 1876 50

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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PMID:Helicobacter pylori UreE, a urease accessory protein: specific Ni(2+)- and Zn(2+)-binding properties and interaction with its cognate UreG. 1947 42

UreG is a GTPase required for assembly of the nickel-containing active site of urease. Herein, a Strep-tagged Klebsiella aerogenes UreG (UreG(Str)) and selected site-directed variants of UreG(Str) were constructed for studying the in vivo effects on urease activation in recombinant Escherichia coli cells, characterizing properties of the purified proteins, and analysis of in vivo and in vitro protein-protein interactions. Whereas the Strep tag had no effect on UreG's ability to activate urease, enzyme activity was essentially abolished in the K20A, D49A, C72A, H74A, D80A, and S111A UreG(Str) variants, with diminished activity also noted with E25A, C28A, and S115A proteins. Lys20 and Asp49 are likely to function in binding/hydrolysis of GTP and binding of Mg, respectively. UreG(Str) binds one nickel or zinc ion per monomer (K(d) approximately 5 microM for each metal ion) at a binding site that includes Cys72, as shown by a 12-fold increased K(d) for nickel ions using C72A UreG(Str) and by a thiolate-to-nickel charge-transfer band that is absent in the mutant protein. Based on UreG homology to HypB, a GTPase needed for hydrogenase assembly, along with the mutation results, His74 is likely to be an additional metal ligand. In vivo pull-down assays revealed Asp80 as critical for stabilizing UreG(Str) interaction with the UreABC-UreDF complex. In vitro pull-down assays demonstrated UreG binding to UreE, with the interaction enhanced by nickel or zinc ions. The metallochaperone UreE is suggested to transfer its bound nickel to UreG in the UreABC-UreDFG complex, with the metal ion subsequently transferring to UreD and then into the nascent active site of urease in a GTP-dependent process.
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PMID:Mutagenesis of Klebsiella aerogenes UreG to probe nickel binding and interactions with other urease-related proteins. 2053 38

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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PMID:Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease. 2123 85

Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence.
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PMID:Wsp1, a GBD/CRIB domain-containing WASP homolog, is required for growth, morphogenesis, and virulence of Cryptococcus neoformans. 2135 79

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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PMID:Chemistry of Ni2+ in urease: sensing, trafficking, and catalysis. 2154 31


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