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PubMed1996

Characterization of the mononickel metallocenter in H134A mutant urease.

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.

Binding Sites↗
PubMed2001

Crystal structure of Klebsiella aerogenes UreE, a nickel-binding metallochaperone for urease activation.

UreE is proposed to be a metallochaperone that delivers nickel ions to urease during activation of this bacterial virulence factor. Wild-type Klebsiella aerogenes UreE binds approximately six nickel ions per homodimer, whereas H144*UreE (a functional C-terminal truncated variant) was previously reported to bind two. We determined the structure of H144*UreE by multi-wavelength anomalous diffraction and refined it to 1.5 A resolution. The present structure reveals an Hsp40-like peptide-binding domain, an Atx1-like metal-binding domain, and a flexible C terminus. Three metal-binding sites per dimer, defined by structural analysis of Cu-H144*UreE, are on the opposite face of the Atx1-like domain than observed in the copper metallochaperone. One metal bridges the two subunits via the pair of His-96 residues, whereas the other two sites involve metal coordination by His-110 and His-112 within each subunit. In contrast to the copper metallochaperone mechanism involving thiol ligand exchanges between structurally similar chaperones and target proteins, we propose that the Hsp40-like module interacts with urease apoprotein and/or other urease accessory proteins, while the Atx1-like domain delivers histidyl-bound nickel to the urease active site.

Animals↗
PubMed1991

Urease (EC 3.5.1.5) inhibition in the sheep rumen and its effect on urea and nitrogen metabolism.

The urease (EC 3.5.1.5) inhibitor, phenylphosphoryldiamidate (PPDA), was given by continuous infusion into the rumen of two sheep nourished by intragastric infusion and into either the rumen or abomasum of four sheep given a pelleted diet containing 119 g crude protein (nitrogen x 6.25)/kg dry matter. PPDA was given at 1 g/d in infusion sheep and 1.5 g/d in the normally-fed sheep. Measurements of urea kinetics were made using single injections of [14C]urea. Urease inhibition was complete within 24 h of starting PPDA infusions to the rumen; in this time-period, urea concentration in rumen contents reached equilibrium with that in plasma and this situation persisted until infusions were terminated. Relative to the control periods, plasma urea and rumen ammonia concentrations were unchanged but urea irreversible loss rate decreased by 26% in infusion sheep and 33% in fed sheep when PPDA was given. Urinary urea excretion was not affected, hence urea degradation, measured by difference, decreased by 77 and 58% respectively in response to urease inhibition. Administration of PPDA to the abomasum resulted in a reduction in rumen urease activity to about 40% of control values but had no effect on urea metabolism. Differences between treatments in daily nitrogen retention were not significant, indicating that under the dietary conditions imposed in these experiments, even substantial changes in urea recycling had only minor effects on the overall N economy of the animal.

Abomasum↗
PubMed2002

Kinetics of inhibition and molecular asymmetry in pigeonpea (Cajanus cajan) urease.

Urease from seeds of pigeonpea showed a time-dependent and irreversible inactivation at very low concentrations of heavy metal ions. Concentration of Cu(2+), Hg(2+) and Ag(+) required for 50% inactivation, on 10 min of incubation, were found to be 2.2 x 10(-6), 2.9 x 10(-8) and 6.3 x 10(-12) M, respectively. The kinetics of inactivation with each of these metal ions was found to be biphasic, with half of the activity being lost in a fast phase and remaining in a slow phase. Acetohydroxamate (AHA) inhibits pigeonpea urease competitively and reversibly with a K(i) of 0.041 mM at pH 7.3. This inhibition was found to be pH dependent. A reversible and time-dependent inhibition was observed with AHA. AHA inhibition revealed biphasic kinetics as observed with the heavy metal ions. Pigeonpea urease was also inhibited by fluoride ions competitively with a K(i) value of 1.23 mM. These inhibition studies suggest the possible interaction of these inhibitors with active site thiol groups and Ni (II) ion. A mechanism has been proposed for each of these inhibitors and compared with inhibition studies reported for other ureases.

Enzyme Inhibitors↗
PubMed2003

Inactivation of jack bean urease by allicin.

Allicin--diallyl thiosulfinate--is the main biologically active component of freshly crushed garlic. Allicin was synthesized as described elsewhere and was tested for its inhibitory ability against jack bean urease in 20 mM phosphate buffer, pH 7.0 at 22 degrees C. The results indicate that allicin is an enzymatic inactivator. The loss of urease activity was irreversible, time- and concentration dependent and the kinetics of the inactivation was biphasic; each phase, obeyed pseudo-first-order kinetics. The rate constants for inactivation were measured for the fast and slow phases and for several concentrations of allicin. Thiol reagents, and competitive inhibitor (boric acid) protected the enzyme from loss of enzymatic activity. The studies demonstrate that urease inactivation results from the reaction between allicin and the SH-group, situated in the urease active site (Cys592).

Binding Sites↗
PubMed2001

Encapsulation of PEG-urease/PEG-AlaDH enzyme system in erythrocyte.

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.

Alanine↗
PubMed1998

Helicobacter pylori urease significantly reduces opsonization by human complement.

The role of Helicobacter pylori urease in opsonization by human complement was investigated. H. pylori wild type strain N6 and isogenic mutants lacking either the large urease subunit (UreB) or an accessory urease protein (UreG) were incubated with different sera. C3b bound to the bacteria was measured by specific staining and flow cytometry. As compared with opsonization of N6 and the UreG-lacking mutant, opsonization of the UreB-lacking mutant was significantly increased after incubation with sera from both H. pylori uninfected (P<.001) or infected (P<.05) persons. However, when sera from uninfected persons were used, effective opsonization of this mutant proved to be dependent mainly on the classical pathway of complement activation. Irrespective of the serum used, opsonization values were very low after selective inactivation of the classical or the alternative pathway. Reduced opsonization of the urease-expressing strains could, to some extent, result from degradation of bound C3b.

Bacterial Proteins↗
PubMed1995

Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis.

Helicobacter pylori, a gram-negative spiral bacterium, is the cause of chronic superficial (type B) gastritis and peptic ulcer disease. The urease enzyme of H. pylori was expressed as an inactive recombinant protein in Escherichia coli, purified as particulate structures of 550-600 kDa molecular mass with a diameter of approximately 12 nm. Given orally, 5 micrograms of urease with an appropriate mucosal adjuvant, such as the labile toxin of E. coli, protected 60%-100% of mice against challenge with virulent Helicobacter felis. Protection correlated with the level of secretory IgA antibodies against urease. Oral administration of antigen was as effective or better than intragastric administration. Parenteral injection of antigen or intragastric administration of high-dose antigen without adjuvant elicited serum IgG but no IgA antibodies and did not confer protection. Recombinant urease as an oral vaccine candidate deserves further investigation as an approach to the prevention of Helicobacter-induced chronic gastroduodenal diseases in humans.

Adjuvants, Immunologic↗
PubMed1975

Inhibition of urease activity by hydroxamic acid derivatives of amino acids.

Hydroxamic acids have been reported to be potent and specific inhibitors of urease (EC 3.5.1.5) activity of plant and bacterial origin. The present investigation was performed on the inhibitory effect of hydroxamic acid derivatives of naturally occurring amino acids on the urease activity of the Jack Bean and the alimentary tracts of rats. Methionine-hydroxamic acid was the most powerful inhibitor (I50=3.9 X 10(-6) M) among nineteen alpha-aminoacyl hydroxamic acids. Phenylalanine-, serine-, alanine-, glycine-, histidine-, threonine-, leucine-, and arginine-hydroxamic acids followed, in order of decreasing inhibitory power. The inhibition proceeded with time at a comparable rate to fatty acyl hydroxamic acid inhibition. The I50 values of alpha-aminoacyl hydroxamic acids were found to be almost equal to those of the corresponding fatty acyl hydroxamic acids. This fact shows that the alpha-amino group did not affect inhibitory power. However, aspartic-beta-, lysine-, and glutamic-gamma-hydroxamic acids, in descending order, were much less inhibitory, probably due to the presence of a carboxyl or omega-amino group. Furthermore, the pH optimum of the inhibition shifted to lower pH in the presence of a carboxyl group, and to a higher pH in e presence of an amino group. The results suggest that the dissociation of an acidic or a basic group reduces the inhibitory power of hydroxamic acid. Hydroxamic acid inhibits urease activity with strict specificity, excpet for aspartic-beta-hydroxamic acid, which inhibited asparaginase competitively. Hydroxamic acid derivatives of amino acids inhibited not only the urease activity of the Jack Bean, but also that of the caecum and ileum parts of the rat intestine.

Amino Acids↗
PubMed1984

Modification of thiol groups of Jack bean urease with diazonium-1H-tetrazole.

Jack bean urease [EC 3.5.1.5] was modified with diazonium-1H-tetrazole (DHT). Reaction of DHT with the enzyme produced a characteristic absorption peak at 320 nm and led to complete loss of the enzymatic activity at a low concentration of DHT. Amino acid analysis of DHT-modified urease showed that only cysteine residues reacted with the reagent and other amino acid residues such as tyrosine, histidine, and lysine did not. The enzymatic activity was protected against DHT-inactivation by the addition of substrate. On the other hand, when the cysteine residues were modified with DHT, the enzyme was not converted to polymeric forms. Furthermore, the binding ability of urease with hydroxamic acid, a specific urease inhibitor, was virtually unaffected by DHT-inactivation. These results indicate that cysteine residues are specifically modified by DHT with concomitant loss of enzymatic activity and polymerization ability, but are not essential for the binding of hydroxamic acid to the enzyme.

Amino Acids↗
PubMed1997

Effect of feeding diets containing an antibiotic, a probiotic, or yucca extract on growth and intestinal urease activity in broiler chicks.

A 6-wk study was conducted to determine the effect of feeding diets containing an antibiotic, a probiotic, or yucca extract on daily gain, feed conversion ratio, and urease activity and ammonia production in intestinal contents of broiler chicks. Four replicates of 10 broiler chicks (average body weight, 48 g) each were assigned to a control or diets containing 0.1% chloroxytetracycline (antibiotic), 0.1% Lactobacillus casei (probiotic), or 0.2% yucca extract. Feeding a diet containing the probiotic significantly (P < 0.05) increased average daily gain during the first 3-wk period compared to the control (30.7 vs 28.7 g). This increase was partly accounted for by increased feed intake. During the first 3 wk, feeding the diet containing probiotic significantly (P < 0.05) decreased urease activity (per gram of collected contents) in small intestinal contents but not in large intestinal contents, compared with the control. Urease activity determined at 6 wk of age was not significantly affected by diet. Our studies indicate that dietary probiotic decreases urease activity in the small intestinal contents of young chicks and thus may be beneficial for improving animal health and growth, especially during early life.

Ammonia↗
PubMed1996

Increased bacterial urease activity in faeces in juvenile chronic arthritis: evidence of altered intestinal microflora?

The intestinal microflora was indirectly evaluated in juvenile chronic arthritis (JCA) by analysing enzyme activities--urease, beta-glucosidase and beta-glucuronidase--in faeces. In 18 out of 26 JCA patients, the illness had been diagnosed during the past year. The control group was composed of eight age-matched control patients and 18 family members of JCA patients (3-36 yr). The mean [95% confidence interval (CI)] urease activity, but not the activities of beta-glucosidase and beta-glucuronidase, in faeces from the JCA group differed from that in the control group: 32.3 (26.6-38.1) nmol/min/mg protein vs 24.0 (16.8-31.6), P = 0.07. The difference was more marked in a comparison of JCA patients with family members (P = 0.03). In a subgroup of subjects, the effect of 10 days oral bacteriotherapy with Lactobacillus GG on faecal enzyme activities was then investigated (n = 8 JCA patients, n = 8 control patients). This short-term oral bacteriotherapy reduced the increased urease activity in faeces of JCA patients. Keeping in mind the small number of subjects, it may be inferred from the present results that the increased urease activity in JCA is specific for the disease, suggesting altered intestinal microflora in JCA.

Administration, Oral↗
PubMed1996

Modified rapid urease test for detection of Helicobacter pylori infection.

BACKGROUND: The rapid urease test is the most widely used standard procedure for the detection of Helicobacter pylori infection. This is because it is a simple, reliable and inexpensive test, which provides results rapidly. AIM: The aim of the present study was the evaluation of a modified rapid urease test (HUT-test) in comparison with the standard CLO-test. In addition, it was questioned whether the performance of the rapid urease test in two biopsy samples from antrum and gastric body would increase the sensitivity of the test. METHODS: One hundred and fifteen consecutive patients undergoing oesophagogastroduodenoscopy were studied. Diagnosis of H. pylori infection was based on histological examination of biopsy samples from antrum and gastric body. The HUT-test was performed in two biopsy specimens from antrum and body, respectively, and the CLO-test in one antral biopsy sample. The time to positivity and the stability of the HUT-test kit at different storage temperatures were previously optimized by adapting the type and concentration of buffer and indicators. RESULTS: Sixty patients (52%) were diagnosed as being infected with H. pylori. Sensitivity of the rapid urease tests in biopsy samples from the antrum was 90% for the HUT-test and 88% for the CLO-test, with a specificity of 100% for both tests. Combining biopsies from the antrum and corpus increased the diagnostic sensitivity of the HUT-test to 93%, but the specificity decreased to 98%. In patients with H. pylori infection, the HUT-test gave positive results more rapidly than the CLO-test (104 +/- 21 min compared with 195 +/- 49 min, respectively, P < 0.02). CONCLUSION: Application of the HUT-test to a simple biopsy from the antrum can establish the diagnosis of H. pylori infection with high accuracy, similar to that of the standard CLO-test. Compared with the CLO-test, the HUT-test has a significantly faster reaction time, which is an obvious clinical advantage. An additional biopsy from the distal gastric body did not add to the diagnostic efficacy in untreated patients, but may be needed in specific circumstances, such as following treatment.

Adult↗
PubMed1990

Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibodies.

The urease of Helicobacter pylori (formerly Campylobacter pylori) has been partly purified by fast protein liquid chromatography. This material contained 10 nm doughnut-like structures when examined by electron microscopy and comprised three major polypeptides (61 kDa, 56 kDa and 28 kDa). Only two of these polypeptides (61 kDa and 28 kDa) were observed in urease-containing material isolated by preparative non-denatured PAGE. Monoclonal antibodies (mAbs) were produced which were directed against two of these polypeptides (56 kDa and 28 kDa). Only mAbs directed against the 28 kDa polypeptide inhibited or captured urease activity. These results suggest that the 56 kDa polypeptide is not essential for enzyme activity. Anti-urease mAbs were used in an indirect immunogold technique to localize the enzyme at the ultrastructural level. In both prefixed bacteria and ultrathin cryosectioned bacteria the enzyme was located on the cell surface and in material apparently shed from that surface.

Antibodies, Monoclonal↗
PubMed1980

The ureases of Proteus strains in relation to virulence for the urinary tract.

The ureases produced by a large number of strains of different Proteus species, some of which were known to have a special affinity for the urinary tract, were examined by polyacrylamide-gel electrophoresis. Each Proteus strain gave a pattern of urease isoenzymes that was characteristic and unique to its species although strains of P. Mirabilis and P. vulgaris gave isoenzyme patterns that were closely similar. There was some minor variation in the patterns of urease isoenzymes even between strains of the same species. This was most noticeable among P. rettgeri strains and to a lesser extent among P. vulgaris strains. No correlation was found between the types of ureases a strain produced and its pathogenicity for the urinary tract.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Crystallization and preliminary X-ray structure determination of jack bean urease with a bound antibody fragment.

Urease allows organisms to use exogenous and internally generated urea as a nitrogen source, by catalyzing the hydrolysis of urea to form ammonia and carbon dioxide. Urease may also participate in the systemic nitrogen-transport pathways and possibly acts as a toxic defence protein. Jack bean urease (JBU) was the first nickel-metalloenzyme identified and was crystallized as early as 1926. Despite this, the structure has not yet been determined. An antibody fragment, Fv, that has a high affinity for JBU has been used to aid crystallization. The complex, which retains full enzyme activity, forms very small crystals that diffract weakly to 3.3 A. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 228.6, c = 130.9 A. The structure of the urease molecule has been solved by molecular replacement using the structure of homogenous enzyme from Klebsiella aerogenes as a search model.

Crystallization↗
PubMed2005

Effect of different organic acids (citric, malic and ascorbic) on intragastric urease activity.

BACKGROUND: The mechanism of citric acid-enhanced Helicobacter pylori urease activity remains unclear. AIM: To compare ascorbic, citric and malic acid given at the same concentration and pH on intragastric urease activity. METHODS: Volunteers received 40 mg of famotidine the evening prior to breath testing. After an overnight fast volunteers were randomized to receive 100 mL of water or 100 mm citric, malic, or ascorbic acid, pH 2.3 containing 75 mg of 13C-urea. At 15 min a second 100 mL solution of one of the test solutions was taken without added urea. RESULTS: Twelve volunteers were studied (eight men, four women, age 19-57, median 50.7) in a randomized-crossover study. The mean breath test result at 30 min with ascorbic (17.5 +/- 5), malic (25.8 +/- 5) and citric acid (29.5 +/- 5) were all significantly greater than with water (9.5 +/- 3). Citric and malic acid were similar (P = 0.699) and significantly greater than ascorbic acid (P < 0.02). When the ascorbic acid was followed by citric acid, the result was similar to that with citrate alone (25.8 +/- 4) and greater than with ascorbic then ascorbic (P = 0.026). CONCLUSIONS: Enhancement of H. pylori urease activity is not strictly a function of the pH. We propose the effect is related to differential effects of the availability of nickel, which is required for urease activity. Citric acid and malic acid were essentially equivalent such that malic acid could substitute for citric acid in the UBT; ascorbic acid would be a poor choice.

Adult↗
PubMed1996

Activation of transcription at divergent urea-dependent promoters by the urease gene regulator UreR.

The Proteus mirabilis and plasmid-encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC-like transcriptional activator. Previously, it was shown that the plasmid-encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea- and UreR-dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid-encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid-encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5 alpha background, as determined by primer-extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR-binding sites within the plasmid-encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid-encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid-encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea.

Bacterial Proteins↗