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)

Bordetella bronchiseptica is a common ureolytic mammalian respiratory pathogen. The urease operon of this organism is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to other urease genes. Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes, nickel donation to the urease active site, and prevention of nickel incorporation until correct formation of the active site, respectively. The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon. UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv. viciae, and may potentially be involved in nickel transport. A transcriptional activator, designated Bordetella bronchiseptica urease regulator (BbuR), is located directly upstream and in the opposite orientation to the urease operon. BbuR shares homology with members of the LysR regulatory protein family. LysR proteins have been shown to regulate urease in Klebsiella aerogenes (NAC), and catalase in Escherichia coli (OxyR), which offers the intracellular bacterium protection from phagolysosome damage. A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was found 27 bp upstream of the urease promoter in B. bronchiseptica. We hypothesise that BbuR controls urease expression which is involved in protection of intracellular B. bronchiseptica from phagolysosomal damage. Comparison of the urease promoter regions of B. bronchiseptica, Bordetella parapertussis ATCC15311 and the urease-negative strain B. pertussis Tohama I revealed no differences in the ureD open reading frame between each species. A cluster of mutations in both B. pertussis and B. parapertussis was found upstream of the urease promoter, in a region proximal to the putative bbuR promoter. The inability of B. pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself.
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PMID:Characterisation of the urease gene cluster in Bordetella bronchiseptica. 952 76

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

Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are synthesized starting from the apoprotein via several intermediates by the action of accessory proteins. The isolation and biochemical characterization of such intermediates is hampered by their low abundance and their lability. Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Sepharose matrix. The tag was fused to the large subunit of [NiFe] hydrogenase 3 (HycE) of Escherichia coli. No significant influence of the affinity peptide on maturation or activity of the protein was observed when the modified gene was integrated into the chromosome by homologous recombination. A tagged nickel-free precursor form of HycE bound quantitatively to a recombinant StrepTactin Sepharose column. More than 90% pure subunit could be obtained after elution with desthiobiotin. The procedure was shown to be more efficient than purification by immobilized metal affinity chromatography using a N-terminal His-tag. General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed.
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PMID:Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis. 960 45

Helicobacter pylori, an important etiologic agent in a variety of gastroduodenal diseases, produces large amounts of urease as an essential colonization factor. We have demonstrated previously that urease is located within the cytoplasm and on the surface of H. pylori both in vivo and in stationary-phase culture. The purpose of the present study was to assess the relative contributions of cytoplasmic and surface-localized urease to the ability of H. pylori to survive exposure to acid in the presence of urea. Toward this end, we compared the acid resistance in vitro of H. pylori cells which possessed only cytoplasmic urease to that of bacteria which possessed both cytoplasmic and surface-localized or extracellular urease. Bacteria with only cytoplasmic urease activity were generated by using freshly subcultured bacteria or by treating repeatedly subcultured H. pylori with flurofamide (1 microM), a potent, but poorly diffusible urease inhibitor. H. pylori with cytoplasmic and surface-located urease activity survived in an acid environment when 5 mM urea was present. In contrast, H. pylori with only cytoplasmic urease shows significantly reduced survival when exposed to acid in the presence of 5 mM urea. Similarly, Escherichia coli SE5000 expressing H. pylori urease and the Ni2+ transport protein NixA, which expresses cytoplasmic urease activity at levels similar to those in wild-type H. pylori, survived minimally when exposed to acid in the presence of 5 to 50 mM urea. We conclude that cytoplasmic urease activity alone is not sufficient (although cytoplasmic urease activity is likely to be necessary) to allow survival of H. pylori in acid; the activity of surface-localized urease is essential for resistance of H. pylori to acid under the assay conditions used. Therefore, the mechanism whereby urease becomes associated with the surface of H. pylori, which involves release of the enzyme from bacteria due to autolysis followed by adsorption of the enzyme to the surface of intact bacteria ("altruistic autolysis"), is essential for survival of H. pylori in an acid environment. The ability of H. pylori to survive exposure to low pH is likely to depend on a combination of both cytoplasmic and surface-associated urease activities.
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PMID:Helicobacter pylori containing only cytoplasmic urease is susceptible to acid. 978 4

Three distinct P type pumps were cloned from H. pylori 69A. Two of these pumps, ATPase 439 and ATPase 948 (CopA), were isolated by gene library screening using DNA oligonucleotide primers. Amino acid similarities found for the predicted proteins were about 50% to Cd2+/Cu2+ pumps. Gene disruption mutagenesis rendered the H. pylori knockout mutants more sensitive to Zn2+ and Cd2+ (ATPase 439) or Cu2+ (CopA). Some of the ATPase 439-deficient mutants were negative for urease activity while the majority of the mutants remained positive. Functional diversity of the pumps was also reflected by the ion affinities found for N-terminal peptides of CopA to Cu2+ and of ATPase 439 to Ni2+, Cu2+ and CO2+. The membrane domain of the two pumps were experimentally shown to consist of eight membrane spans. When ATPase 439 was expressed under control of a tac promoter in Escherichia coli, vanadate-sensitive phosphate accumulation was observed cytochemically along the membrane of the host cells. The third P type pump (ATPase 115) which also exhibited homology to transition metal ATPase was identified by sequencing a library of H. pylori membrane genes. The hydropathy plot of this pump was very similar to the former H. pylori ATPases whereas the N-terminal ion binding region was distinct. It was concluded that, in H. pylori, the presence of three transition metal ATPases with distinct ion specificity contributes to the adaptive mechanisms for gastric survival.
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PMID:Properties and function of the P type ion pumps cloned from Helicobacter pylori. 978 54

Since 1995, crystal structures have been determined for many transition-metal enzymes, in particular those containing the rarely used transition metals vanadium, molybdenum, tungsten, manganese, cobalt and nickel. Accordingly, our understanding of how an enzyme uses the unique properties of a specific transition metal has been substantially increased in the past few years. The different functions of nickel in catalysis are highlighted by describing the active sites of six nickel enzymes - methyl-coenyzme M reductase, urease, hydrogenase, superoxide dismutase, carbon monoxide dehydrogenase and acetyl-coenzyme A synthase.
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PMID:Active sites of transition-metal enzymes with a focus on nickel. 991 55

It has been shown that urea in fermented beverages and foods can serve as a precursor of ethylcarbamate, a potential carcinogen, and acid urease is an effective agent for removing urea in such products. We describe herein the purification and characterization of a novel acid urease from Arthrobacter mobilis SAM 0752 and show its unique application for the removal of urea from fermented beverages using the Japanese rice wine, sake, as an example. The purified acid urease showed an optimum pH for activity at pH 4.2. The enzyme exhibited an apparent K(m) for urea of 3.0 mM and a Vmax of 2370 mumol of urea per mg and min at 37 degrees C and pH 4.2. Gel permeation chromatographic and sodium dodecyl sulfate gel electrophoretic analyses showed that the enzyme has an apparent native molecular weight (M(r)) of 290,000 and consisted of three types of subunit proteins (M(r), 67,000, 16,600, 14,100) denoted by alpha, beta, and gamma. The most probable stoichiometry of the subunits was estimated to be alpha: beta: gamma = 1:1:1, suggesting the enzyme subunit structure of (alpha beta gamma)3. The enzyme also existed as an aggregated form with an M(r) of 580,000. The purified enzyme contained 2 g-atom of nickel per alpha beta gamma unit of the enzyme. Enzyme activity was inhibited by acetohydroxamic acid, HgCl2, and CuCl2. The isoelectric point of the native enzyme was estimated by gel electrofocusing to be 6.8. Urea (50 ppm), which was exogenously added to sake (pH 4.4, 17 +/- 1% (v/v) ethanol), was completely decomposed by incubation with the enzyme (0.09 U ml-1) at 15 degrees C for 13 days. The enzyme was unstable at temperatures higher than 65 degrees C and pHs lower than 4, and was completely inactivated under the conditions of a pasteurization step involved in the traditional sake-making processes. These results indicate that the enzyme is applicable to the elimination of urea in fermented beverages with minimal modification to the conventional process.
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PMID:Purification, characterization, and application of an acid urease from Arthrobacter mobilis. 1019 59

Helicobacter pylori urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH3 and CO2. We sought to identify H. pylori genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both H. pylori urease and the NixA nickel transporter. Escherichia coli SE5000 and DH5alpha transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An H. pylori DNA library was cotransformed into SE5000 (pHP8080) and DH5alpha (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained hp0548, a gene that potentially encodes a DNA helicase found within the cag pathogenicity island, and hp0511, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in E. coli (pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the flbA (also known as flhA) flagellar biosynthesis/regulatory gene (hp1041), an lcrD homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in E. coli (pHP8080) containing the subcloned flbA gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in E. coli (pHP8080) containing the flbA gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in H. pylori. These results suggest that H. pylori genes may modulate urease activity.
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PMID:Isolation of Helicobacter pylori genes that modulate urease activity. 1019 12

Expression of the active urease of the enterobacterium, Klebsiella aerogenes, requires the presence of the accessory genes (ureD, ureE, ureF, and ureG) in addition to the three structural genes (ureA, ureB, and ureC). These accessory genes are involved in functional assembly of the nickel-metallocenter for the enzyme. Characterization of ureF gene has been hindered, however, since the UreF protein is produced in only minute amount compared to other urease gene products. In order to overexpress the ureF gene, a recombinant pMAL-UreF plasmid was constructed from which the UreF was produced as a fusion with maltose-binding protein. The MBP-UreF fusion protein was purified by using an amylose-affinity column chromatography followed by an anion exchange column chromatography. Polyclonal antibodies raised against the fusion protein were purified and shown to specifically recognize both MBP and UreF peptides. The UreF protein was shown to be unstable when separated from MBP by digestion with factor Xa.
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PMID:Expression of the recombinant Klebsiella aerogenes UreF protein as a MalE fusion. 1040 30

In order to model the active site of urease which contains two nickel ions with differing coordination geometries new parameters were derived for the AMBER* force field. These parameters were obtained by structure based optimization and use a single set of parameters with points on a sphere approach to model nickel(II) high-spin in all its coordination geometries. The force field was successfully used to model the active site of urease and to predict that a bridging water between the two nickel ions in urease was missing from the solid state structure of urease. A thorough conformational search was undertaken to find the conformations available to urea within urease. All the low energy conformations found were used to determine a consensus urea binding model.
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PMID:A molecular mechanical analysis of the active site of urease with a special emphasis on determining the binding conformations available to oxygen-bound urea. 1049 27


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