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)

Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess 15N, that is the excess enrichment of 15N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. 15N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of 15N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of 15N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with 15N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the 15N label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.
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PMID:Helicobacter pylori utilises urea for amino acid synthesis. 882 3

In vivo urease metallocenter assembly in Klebsiella aerogenes requires the presence of several accessory proteins (UreD, UreF, and UreG) and is further facilitated by UreE. In this study, UreG was isolated and shown to be a monomer with an Mr of 21,814 +/- 20 based on gel filtration chromatography and mass spectrometric results. Although it contains a P-loop motif typically found in nucleotide-binding proteins, UreG did not bind or hydrolyze ATP or GTP, and it exhibited no affinity for ATP- and GTP-linked agarose resins. Site-directed mutagenesis of ureG allowed the substitution of Ala for Lys-20 or Thr-21 in the P-loop motif and resulted in the production of inactive urease in cells grown in the presence of nickel; hence, an intact P-loop may be essential for UreG to function in vivo. These mutant cells were unable to synthesize the UreD-UreF-UreG-urease apoprotein species that are thought to be the key urease activation complexes in the cell. An insoluble protein species containing UreD, UreF, and UreG (termed the DFG complex) was detected in cells carrying deletions in ureE and the urease structural genes. The DFG complex was solubilized in 0.5% Triton X-100 detergent, shown to bind to an ATP-linked agarose resin, and found to elute from the resin in the presence of Mg-ATP. In cells containing a UreG P-loop variant, the DFG complex was formed but did not bind to the nucleotide-linked resin. These results suggest that the UreG P-loop motif may be essential for nucleotide binding by the DFG complex and support the hypothesis that nucleotide hydrolysis is required for in vivo urease metallocenter assembly.
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PMID:Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease. 920 19

The isolation of a new motile, gram-negative, heterotrophic, sulfur-reducing, microaerophilic, vibrioid bacterium, strain F1F6, from oxidized marine surface sediment (Arcachon Bay, French Atlantic coast) is described. Hydrogen (with acetate as the carbon source), formate (with acetate as the carbon source), pyruvate, lactate, alpha-ketoglutarate, glutarate, glutamate, and yeast extract supported growth with elemental sulfur under anaerobic conditions. Apart from H2 and formate, the oxidation of the substrates was incomplete. Microaerophilic growth was supported with hydrogen (acetate as the carbon source), formate (acetate as the carbon source), acetate, propionate, pyruvate, lactate, alpha-ketoglutarate, glutamate, yeast extract, fumarate, succinate, malate, citrate, and alanine. The isolate grew fermentatively with fumarate, succinate being the only organic product. Elemental sulfur and oxygen were the only electron acceptors used. Vitamins or amino acids were not required. The isolate was oxidase, catalase, and urease positive. Comparative 16S rDNA sequence analysis revealed a tight cluster consisting of the validly described species Sulfurospirillum deleyianum and the strains SES-3 and CCUG 13942 as the closest relatives of strain F1F6 (level of sequence similarity, 91.7 to 92.4%). Together with strain F1F6, these organisms form a novel lineage within the epsilon subclass of proteobacteria clearly separated from the described species of the genera Arcobacter, Campylobacter, Wolinella, and Helicobacter. Due to the phenotypic characteristics shared by strain F1F6 and S. deleyianum and considering their phylogenetic relationship, we propose the inclusion of strain F1F6 in the genus Sulfurospirillum, namely, as S. arcachonense sp. nov. Based on the results of this study, an emended description of the genus Sulfurospirillum is given.
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PMID:Sulfurospirillum arcachonense sp. nov., a new microaerophilic sulfur-reducing bacterium. 933 31

Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis.
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PMID:Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand. 955 61

Mutation in Eu3 eliminates activity of both soybean ureases, the embryo-specific (encoded by Eu1) and the tissue-ubiquitous (encoded by Eu4). eu3-e1 is a completely recessive null allele. Eu3-e3 is a semi-dominant specifying 0.1% wild-type urease activity in the homozygous state and 5-10% as a heterozygote (Meyer-Bothling et al. 1987). Antibodies to plant UreG, a homologue of the bacterial urease accessory protein, revealed a 32 kDa protein (p32) in embryos of the Eu3/Eu3 precursor genotype. p32 is identical to UreG by the criteria of size, antigenicity, and its ability to bind Ni2+, a trait expected from the deduced histidine-rich N-terminus of UreG. UreG was absent in eu3-e1/eu3-e1, and lack of UreG co-segregated with eu3-e1. Eu3-e3 specified a UreG transcript which coded valine in place of alanine at residue 142 (A142V) confirming thatEu3 encodes UreG, which is renamed Eu3. Eu3 (A142V) retained Ni-binding ability. Eu3 is directly involved in urease activation, since anti-Eu3 (UreG) antibodies inhibited the in vitro activation of urease. Eu1 (embryo urease) and Eu3 accumulated in parallel in the developing embryo. The presence of Eu1 was not necessary for the high embryonic level of Eu3. However, the presence of Eu3 appeared to be important for accumulation of Eu1, perhaps by stabilizing it by Ni insertion. At the level of sensitivity employed Eu3 was detected in crude extracts of embryos but not non-embryonic tissues which have 1/500th the embryo urease activity. Functional Eu3, however, is necessary for activation of the ubiquitous urease in non-embryonic tissues.
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PMID:The soybean Eu3 gene encodes an Ni-binding protein necessary for urease activity. 1065 50

Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 degrees C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH4(+)-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein(-1) x min(-1)) and low deaminating (21 nmol x mg protein(-1) x min(-1)) activities. Its Km-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deamination about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg protein(-1) x min(-1)). Its affinity for glutamate was poor (Km = 23.7 mM), while that for the NH4+ replacing NH2OH was high (Km = 0.19 mM). pH-optimum was found at 7.0. Glutamate synthase (= GOGAT) revealed similar low activity (62 nmol x mg protein(-1) x min(-1)), Km-values for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate (Km = 2.55 mM) and for glutamate (Km = 3.13 mM), but clearly different Km-values for 2-oxoglutarate (1.46 mM) and for oxaloacetate (0.13 mM). Activity at optimum pH of about 8.0 was 506 nmol x mg protein(-1) x min(-1) for aspartate conversion, but only 39 nmol x mg protein(-1) x min(-1) at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg protein(-1) x min(-1)), substrate affinities (Km for alanine = 6.30 mM, for 2-oxoglutarate = 0.45 mM) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribution of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nmol x mg protein(-1) x min(-1)) whose substrate affinity was rather high (Km = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed.
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PMID:Investigations into enzymes of nitrogen metabolism of the ectomycorrhizal basidiomycete, Suillus bovinus. 1081 9

No intravenously injectable enzyme preparate containing urease as an alternetive to hemodialysis, hemoperfusion and CAPD systems in patients having chronic renal failure has been encountered in literature. In this study, it has been aimed to convert blood urea to alanine by using PEG-urease/PEG-AlaDH enzyme pair encapsulated within living erythrocyte. In this system, urea is decomposed into NH3 and HCO3- and the ammonia released is converted into alanine by reacting pyruvate under the catalytic action of alaninedehydrogenase. The production of pyruvate and NADH by erythrocyte required in the second stage of the reaction will make the process a feasible and ceaseless one. The success of the system will enable the renal patients with diabetes mellitus. Urease and AlaDH were covalently immobilized on activated PEG. PEG-urease/PEG-AlaDH were encapsulated in erythrocyte (1/1)(v/v) by using slow dialysis methods. The activity of enzyme system, encapsulation yield and hemogram analysis were determined for each sample.
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PMID:Encapsulation of PEG-urease/PEG-AlaDH enzyme system in erythrocyte. 1170 64

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. The nickel permease NixA of H. pylori is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79-both part of the characteristic sequence motif-and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E. coli system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.
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PMID:Conserved low-affinity nickel-binding amino acids are essential for the function of the nickel permease NixA of Helicobacter pylori. 1184 75

In our system, urease/AlaDH have been encapsulated within erythrocytes by using slow dialysis methods. Urea is decomposed into ammonia and bicarbonate and the ammonia released is converted into alanine by reacting pyruvate under the catalytic action of AlaDH. It is very important for our that products are formed quickly but the ammonia is not connected definetely. For this aim, urease/AlaDH we encapsulated using different enzyme activity ratio (0.5:1.5; 0.5:2.5; 0.25:1.25 U/U urease/AlaDH). The activities of enzyme systems, encapsulation yield, McV, McH, and McHc were measured for each sample. Investigated results suggest that loaded enzyme systems can be used as potential carrier systems for the removal of high levels of urea from blood.
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PMID:In vitro study of urease/AlaDH enzyme system encapsulated into human erythrocytes and research into its medical applications. 1200 Feb 28

Identification of the environmental triggers involved in the expression of virulence genes is a fundamental objective in studies of bacterial pathogens. For uropathogens, urea, found in the urinary tract at concentrations of up to 500 mm, functions as an environmental signal. Urea freely diffuses into the bacterium Providencia stuartii and activates UreR, a member of the AraC family of transcriptional activators. Active UreR promotes transcription of virulence-associated urease genes and alerts the organisms of its immediate milieu. Thus, the UreR.urea complex has a dual role, acting as both a transcriptional activator as well as an environmental sensor. Here, we describe the molecular events associated with activation of gene expression by urea-bound UreR. The K(d) of the urea.UreR binding reaction was measured as 0.2 mm by fluorescence quenching assays, and the shape of the binding curve indicated a single specific urea-binding site on UreR. Histidine residues are critical for urea binding in urease, and therefore to identify the urea-binding site in UreR, five mutant UreR forms were generated with histidine to alanine substitutions. Two of the mutants (UreR(c)) exhibited a constitutive phenotype by both activating transcription and binding to DNA with an increased affinity in the absence of urea. The UreR(c) bound urea with an affinity similar to that of wild-type UreR. We concluded, therefore, that the mutations resulting in constitutive activity were not involved in the UreR.urea interaction. UreR was activated, then, either by binding urea or by histidine to alanine substitutions at one of two positions. Circular dichroism indicated little change in the structure of UreR when activated, and size-exclusion chromatography demonstrated that both rUreR and rUreR(c) were dimers in both the presence and absence of urea. Thus, the structural changes associated with activation are subtle.
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PMID:Urea-dependent signal transduction by the virulence regulator UreR. 1214 87


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