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:3.5.1.5 (urease)
7,257 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interaction of beta-mercaptoethanol with urease produces large, rapid and fully reversible spectral changes in that part of the electronic absorption spectrum which is associated with the tightly bound nickel ions. The spectrophotometrically determined value of the dissociation constant of the beta-mercaptoethanol-urease complex (0.9 +/- 0.05 mM at pH 7.12 and 25 degrees C) is in agreement with the Ki (0.72 +/- 0.26 mM) for beta-mercaptoethanol acting as a competitive inhibitor in the hydrolysis of urea. This constitutes direct evidence that the nickel in jack bean urease is at the active site. Inhibition of urease by phosphoramidate is slowly achieved and slowly reversed, and upon reactivation of the isolated phosphoramidate-urease complex, phosphoramidate is regenerated in good yield. Spectrophotometric experiments indicate that phosphoramidate binds to nickel ion in urease. Competition with beta-mercaptoethanol was used to determine a dissociation constant (1.23 +/- 0.10 mM at pH 7.12 and 25 degrees C) for a fluoride-evidence is presented which indicates that in the presence of urea, a ternary complex (fluoride-urea-urease) is formed.
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PMID:Jack bean urease (EC 3.5.1.5). III. The involvement of active-site nickel ion in inhibition by beta-mercaptoethanol, phosphoramidate, and fluoride. 727 36

Urease (urea amidohydrolase; EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbamate. The latter compound spontaneously decomposes to yield another molecule of ammonia and carbonic acid. The urease phenotype is widely distributed across the bacterial kingdom, and the gene clusters encoding this enzyme have been cloned from numerous bacterial species. The complete nucleotide sequence, ranging from 5.15 to 6.45 kb, has been determined for five species including Bacillus sp. strain TB-90, Klebsiella aerogenes, Proteus mirabilis, Helicobacter pylori, and Yersinia enterocolitica. Sequences for selected genes have been determined for at least 10 other bacterial species and the jack bean enzyme. Urease synthesis can be nitrogen regulated, urea inducible, or constitutive. The crystal structure of the K. aerogenes enzyme has been determined. When combined with chemical modification studies, biophysical and spectroscopic analyses, site-directed mutagenesis results, and kinetic inhibition experiments, the structure provides important insight into the mechanism of catalysis. Synthesis of active enzyme requires incorporation of both carbon dioxide and nickel ions into the protein. Accessory genes have been shown to be required for activation of urease apoprotein, and roles for the accessory proteins in metallocenter assembly have been proposed. Urease is central to the virulence of P. mirabilis and H. pylori. Urea hydrolysis by P. mirabilis in the urinary tract leads directly to urolithiasis (stone formation) and contributes to the development of acute pyelonephritis. The urease of H. pylori is necessary for colonization of the gastric mucosa in experimental animal models of gastritis and serves as the major antigen and diagnostic marker for gastritis and peptic ulcer disease in humans. In addition, the urease of Y. enterocolitica has been implicated as an arthritogenic factor in the development of infection-induced reactive arthritis. The significant progress in our understanding of the molecular biology of microbial ureases is reviewed.
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PMID:Molecular biology of microbial ureases. 756 14

Helicobacter pylori urease is a nickel-containing enzyme that hydrolyzes urea to bicarbonate and ammonia. Andrews et al. (J. Am. Chem. Soc. 1986, 108, 7124) have shown that amides and esters of phosphoric acid are slow, tight-binding inhibitors of urease isolated from jack bean. We show that 4-substituted phenyl phosphorodiamidates (4-R-PhOP(=O)(NH2)2) are slow-binding inhibitors of H. pylori urease with no evidence of kinetic saturation. Their second-order rates of inhibition ki are strongly correlated with phenol pKa (e.g. R = NO2, ki = 2.5 x 10(5) M-1s-1; R = OMe, ki = 1.2 x 10(4) M-1s-1). The Bronsted beta for inhibition is 0.4, similar to that of model system SN2(P) reactions. Based on these observations, we suggest that urease inhibition is covalent but reversible, involving a common phosphoacyl enzyme intermediate.
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PMID:Inhibition of Helicobacter pylori urease by phenyl phosphorodiamidates: mechanism of action. 764 8

Urease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastritis and peptic ulceration. This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea. When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structural subunits are overexpressed, exogenous NiCl2 is added, and the host strain is grown in medium that does not chelate free Ni2+. As wild-type H. pylori does not require such conditions for very high levels of urease expression, we reasoned that additional genes were required to accumulate the metal ion. To isolate such genes, E. coli SE5000 (pHP808), which carries the H. pylori urease gene cluster, was complemented with a lambda ZAP-derived plasmid library of the H. pylori chromosome. One of 1000 ampicillin-resistant clones, plated onto urea segregation agar, produced detectable urease. Urease activity of this co-transformant, grown in Luria broth containing 1 microM NiCl2, was 36 mumol NH3 min-1 mg-1 protein. Urease-enhancing activity, which is not directly linked to the urease gene cluster, was localized by subcloning and nucleotide sequencing. The largest open reading frame, designated nixA, predicted a polypeptide of 34,317 Da that displayed characteristics of an integral membrane protein. In vitro transcription-translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size. An in-frame Bal31 deletion within nixA abolished urease-enhancing activity. At 50 nM NiCl2, E. coli containing the nixA clone transported 1250 +/- 460 pmol Ni2+ min-1 10(-8) cells, whereas the vector control transported only 140 +/- 85 pmol Ni2+ min-1 10(8) cells, i.e. significantly less (P = 0.01). We conclude that NixA confers upon E. coli a high-affinity nickel-transport system (KT = 11.3 +/- 2.4 nM; Vmax = 1750 +/- 220 pmol Ni2+ min-1 10(-8) cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.
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PMID:Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions. 765 Nov 42

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (pILL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (pILL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (MBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.
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PMID:Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity. 771 57

In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.
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PMID:Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation. 772 85

The crystal structure of urease from Klebsiella aerogenes has been determined at 2.2 A resolution and refined to an R factor of 18.2 percent. The enzyme contains four structural domains: three with novel folds playing structural roles, and an (alpha beta)8 barrel domain, which contains the bi-nickel center. The two active site nickels are 3.5 A apart. One nickel ion is coordinated by three ligands (with low occupancy of a fourth ligand) and the second is coordinated by five ligands. A carbamylated lysine provides an oxygen ligand to each nickel, explaining why carbon dioxide is required for the activation of urease apoenzyme. The structure is compatible with a catalytic mechanism whereby urea ligates Ni-1 to complete its tetrahedral coordination and a hydroxide ligand of Ni-2 attacks the carbonyl carbon. A surprisingly high structural similarity between the urease catalytic domain and that of the zinc-dependent adenosine deaminase reveals a remarkable example of active site divergence.
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PMID:The crystal structure of urease from Klebsiella aerogenes. 775 94

Helicobacter pylori is a human gastrointestinal pathogen involved in gastritis, duodenal ulcers, and gastric neoplasia. This microorganism produces large amounts of a urease which, like all known ureases, has nickel in the active site. We have identified a protein in clinical isolates of H. pylori and an identical protein in the ferret pathogen Helicobacter mustelae that strongly binds Ni2+ and Zn2+. This protein has been named Hpn to emphasize its origins in H. pylori and its affinity for nickel. The encoding hpn gene, cloned and expressed in Escherichia coli ER1793, has an open reading frame (180 bp) that specifies a protein with a calculated molecular mass of 7,077 Da and with the same amino-terminal sequence as that of wild-type Hpn. The deduced sequence of Hpn consists of 60 amino acids, of which 28 (47%) are histidines. The hpn gene does not map with the urease gene cluster on the H. pylori chromosome. An Hpn-negative, isogenic H. pylori strain, generated by hpn gene deletion and grown on blood agar, had the same urease activity that wild-type cells did. Thus, the role of Hpn in helicobacters is unknown.
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PMID:Protein Hpn: cloning and characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and Helicobacter mustelae. 779 85

Assembly of protein metallocenters is not well understood. Urease offers a tractable system for examination of this process. Formation of the urease metallocenter in vivo is known to require four accessory proteins: UreD, postulated to be a urease-specific molecular chaperone; UreE, a nickel(II)-binding protein; and UreF and UreG, of unknown function. Activation of purified Klebsiella aerogenes urease apoprotein was accomplished in vitro by providing carbon dioxide (half-maximal activation at approximately 0.2 percent carbon dioxide) in addition to nickel ion. Activation coincided with carbon dioxide incorporation into urease in a pH-dependent reaction (pKa > or = 9, where Ka is the acid constant). The concentration of carbon dioxide also affected the amount of activation of UreD-urease apoprotein complexes. These results suggest that carbon dioxide binding to urease apoprotein generates a ligand that facilitates productive nickel binding.
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PMID:Requirement of carbon dioxide for in vitro assembly of the urease nickel metallocenter. 785 93

HoxN, an integral membrane protein with seven transmembrane helices and a molecular mass of 33.1 kDa, is involved in high-affinity nickel transport in Alcaligenes eutrophus H16. From genetic analyses, it has been concluded that HoxN is a single-component ion carrier. To investigate this assumption, hoxN was introduced into Escherichia coli. The recombinant strain showed significantly enhanced nickel uptake in a short-interval assay. Likewise, growth in the presence of 63NiCl2 yielded a more than 15-fold-increased cellular nickel content. The HoxN-based nickel transport activity could also be demonstrated in a physiological assay: an E. coli strain coexpressing hoxN and the urease operon of Klebsiella aerogenes exhibited urease activity 10-fold greater than that in the strain lacking a functional hoxN. These results strongly suggest that HoxN is sufficient to operate as a nickel permease. Multiple sequence alignment of HoxN and four other bacterial membrane proteins implicated in nickel metabolism revealed two conserved signatures which may play a role in the nickel translocation process.
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PMID:The Alcaligenes eutrophus protein HoxN mediates nickel transport in Escherichia coli. 789 9


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