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)

Urease was purified (4126-fold) from Aspergillus niger (NRRL 003) to a homologous enzyme preparation with a specific activity of 1341 mumol min-1 (mg protein)-1. One species of urease was detected in A. niger, with Km = 3.0 mM, native molecular mass 250,000 Da, pH optimum of 8.0 and a high specificity for urea. Hydroxyurea was a strong competitive inhibitor of urease activity, while N-methylurea acted as a weak uncompetitive inhibitor, based on Lineweaver-Burk and Eadie-Hoftstee plots. The activity of urease was enhanced by, but not dependent on, the presence of Na2EDTA, DL-dithiothreitol (< or = 0.1 to 5.0 mM), Ca2+, Ba2+ and citrate (2 to 20 mM). Urease activity was not affected by Na+, K+, Cl-, Br-, acetate or nitrate (2 to 20 mM), but was significantly decreased in the presence of Li+, Ni2+, Mg2+, Zn2+ or I-. Urease activity decreased 26.0% after 30 min at 65 degrees C, and 86.5% and 100.0% after 5 and 1 min at 80 and 100 degrees C, respectively. Urease activity decreased 30.5% after 90 d at 4 degrees C and 21.0% after 28 d at -20 or -80 degrees C.
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PMID:Isolation and characterization of urease from Aspergillus niger. 833 11

Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni2+ into apoenzyme.
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PMID:Proteus mirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme. 850 Aug 94

The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli. Enzymatic activity, however, has been very weak compared with that in clinical isolates of H. pylori. Conditions under which near wild-type urease activity was achieved were developed. E. coli. SE5000 containing recombinant H. pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H. pylori UMAB41 (100 U/mg of protein), from which the genes were cloned. Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine. In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein. As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids. We conclude that recombinant H. pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided.
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PMID:Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli. 850 Aug 93

Helicobacter pylori urease belongs to a family of highly conserved urea-hydrolyzing enzymes. A common feature of these enzymes is the presence of two Lewis acid nickel ions and a reactive cysteine residue in the active site. The H+/K(+)-ATPase inhibitor omeprazole is a prodrug of a sulfenamide which covalently modifies cysteine residues on the luminal side of the H+/K(+)-ATPase of gastric parietal cells. Omeprazole and eight analogues were selected based on their chemical, electronic, and kinetic properties, and each was incubated with viable H. pylori in phosphate-buffered saline at pH 7.4 for 30 min, after which 100 mM urea was added and the amount of ammonia formed analyzed after a further 10 min. Inhibition between 0% and 100% at a 0.1 mM concentration was observed for the different analogues and could be expressed as a function of the pKa-value of the pyridine, the pKa-value of the benzimidazole, the overall lipophilicity, and, most importantly, the rate of sulfenamide formation, in a quantitative structure-activity relationship. The inhibition was potentiated by a lower pH (favoring the formation of the sulfenamide) but abolished in the presence of beta-mercaptoethanol (a scavenger of the sulfenamide). Structural analogues incapable of yielding the sulfenamide did not inhibit ammonia production. Treatment of Helicobacter felis-infected mice with 230 mumol/kg flurofamide b.i.d. for 4 weeks, known to potently inhibit urease activity in vivo, as a means of eradicating the infection, was tested and compared with the effect of 125 mumol/kg omeprazole b.i.d. for 4 weeks. Neither treatment proved efficacious.
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PMID:Structure-activity relationship of omeprazole and analogues as Helicobacter pylori urease inhibitors. 852 4

Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration in humans, synthesizes urease, a nickel metalloenzyme, as its most abundant protein. NixA, a high-affinity nickel transport protein, allows synthesis of catalytically active urease when coexpressed with H. pylori urease in an Escherichia coli host. To determine whether NixA is essential for the production of active urease in H. pylori, nixA was insertionally inactivated with a kanamycin resistance cassette (aphA) and this construct was electroporated into H. pylori ATCC 43504; allelic exchange mutants were selected on kanamycin-containing medium. The nixA mutation, confirmed by PCR, reduced urease activity by 42% (140 +/- 70 micromol of NH3/min/mg of protein in the mutant versus 240 +/- 100 micromol of NH3/min/mg of protein in the parent (P = 0.037). Rates of nickel transport were dramatically reduced (P = 0.0002) in the nixA mutant (9.3 +/- 3.7 pmol of Ni2+/min/10(8) bacteria) of H. pylori as compared with the parent strain (30.2 +/- 8.1 pmol of Ni2+/min/10(8) bacteria). We conclude that NixA is an important mediator of nickel transport in H. pylori. That residual nickel transport and urease activity remain in the nixA mutant, however, provides evidence for the presence of a redundant transport system in this species.
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PMID:Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity. 869 29

A mutant form of Klebsiella aerogenes urease possessing Ala instead of His at position 134 (H134A) is inactive and binds approximately half the normal complement of nickel (Park, I.-S., and Hausinger, R. P.(1993) Protein Sci. 2, 1034-1041). The crystal structure of the H134A protein was obtained at 2.0-A resolution, and it confirms that only Ni-1 of the two nickel ions found in the native enzyme is present. In contrast to the pseudotetrahedral geometry observed for Ni-1 in native urease (where it is liganded by His-246, His-272, one oxygen atom of carbamylated Lys-217, and a water molecule at partial occupancy), the mononickel metallocenter in the H134A protein was found to possess octahedral geometry and was coordinated by the above protein ligands plus three water molecules. The nickel site of H134A urease was probed by UV-visible, variable temperature magnetic circular dichroism, and x-ray absorption spectroscopies. The spectroscopic data are consistent with the presence of Ni(II) in octahedral geometry coordinated by two histidylimidazoles and additional oxygen and/or nitrogen donors. These data underscore the requirement of Ni-2 for formation of active urease and demonstrate the important role of Ni-2 in establishing the proper Ni-1 coordination geometry.
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PMID:Characterization of the mononickel metallocenter in H134A mutant urease. 870 15

X-ray absorption spectroscopy (XAS) has been applied to urease from Bacillus pasteurii, a highly ureolytic soil bacterium, with the aim of elucidating the structural details of the nickel-containing active site. The results indicate the presence of octahedrally coordinated Ni2+, in a sphere of six N/O donors at an average distance of 0.203 nm. An average of two histidine residues are bound to nickel. The experimental evidence suggests direct binding of the urease inhibitor phenylphosphorodiamidate to Ni2+. These spectroscopic results are in agreement with previous findings on both plant and microbial ureases, but differ in some respect from the results obtained by X-ray crystallography analysis of Klebsiella aerogenes urease.
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PMID:X-ray absorption spectroscopy study of native and phenylphosphorodiamidate-inhibited Bacillus pasteurii urease. 870 19

Urease from Klebsiella aerogenes [Jabri et al. (1995) Science 268, 998-1004] is an (alpha beta gamma)3 trimer with each alpha-subunit having an (alpha beta)8-barrel domain containing a binickel active center. Here we examine structure-function relations for urease in more detail through structural analysis of the urease apoenzyme at 2.3 A resolution and mutants of two key catalytic residues (H219A and H320A) at 2.5 A resolution. With the exception of the active site, in which a water molecule takes the place of the missing carbamate and nickel atoms, the structure of the apoenzyme is nearly identical to that of the holoenzyme, suggesting a high degree of preorganization which helps explain the tight binding of nickel. In the structure of H219A, the major change involves a conformational shift and ordering of the active site flap, but a small shift in the side chain of Asp alpha 221 could contribute to the lower activity of H219A. In the H320A structure, the catalytic water, primarily a Ni-2 ligand in the holoenzyme, shifts into a bridging position. This shift shows that the nickel ligation is rather sensitive to the environment and the change in ligation may contribute to the 10(5)-fold lower activity of H320A. In addition, these results show that urease is resilient to the loss of nickel ions and mutations. Analysis of the urease tertiary/quaternary structure suggests that the stability of this enzyme may be largely due to its burial of an unusually large fraction of its residues: 50% in the gamma-subunit, 30% in the beta-subunit, and 60% in the alpha-subunit.
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PMID:Structures of the Klebsiella aerogenes urease apoenzyme and two active-site mutants. 871 50

As in any other bacterium, Helicobacter pylori synthesizes two heat shock proteins, the HspA (GroES or Hsp 10 homologue) and the HspB (GroEL or Hsp60 homologue). This article summarizes the present knowledge of genetics, function and the antigenic, immunogenic and protective properties of these two abundant proteins. H. pylori HspA and HspB antigens have vital functions for the bacterium; they share most of the bacterial chaperonin characteristics. However, the unique structure of HspA and its unique capacity to specifically bind nickel ions, strongly suggest an essential role of HspA with regard to the urease metallo-enzyme. The putative role of the H. pylori Hsp antigens in autoimmunity is also addressed.
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PMID:Heat shock proteins of Helicobacter pylori. 873 Feb 59

Helicobacter pylori produces a 550 kDa, multimeric, nickel-containing urease that catalyses the hydrolysis of urea to yield ammonia and carbonic acid. The ure gene cluster, comprised of seven genes, encodes the two structural subunits UreA (26.5 kDa) and UreB (60.3 kDa), and five accessory proteins: UreI, UreE, UreF, UreG and UreH. Accessory proteins are required for nickel ion insertion into the apoenzyme. The native protein consists of six copies each of UreA and UreB; two nickel ions are coordinated into each UreB active site. Urease is found in the cytosol, but may also localize on the surface (although this may be an artefact) and elicits a strong serum immunoglobulin response. Urease aids in colonization of the host by neutralizing gastric acid and providing ammonia for bacterial protein synthesis. Host defences are avoided by urease by continuing to neutralize acid locally and by shedding urease, which may be bound by immunoglobulin, from the surface of the bacterium. Host tissues can be damaged directly by the urease-mediated generation of ammonia and indirectly by urease-induced stimulation of the inflammatory response, including recruitment of leukocytes and triggering of the oxidative burst in neutrophils.
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PMID:The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic ulceration. 873 Feb 60


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