Repression of urease biosynthesis in Neurospora crassa by ammonium ions]

The regulation of the synthesis of the enzyme urease (urea amido hydrolase E.C. 3.5.1.5.) in Neurospora crassa was investigated. The biosynthesis of urease is repressed by ammonium ions. Under ammonium excess conditions the specific activity of urease decreases from 0.980 to 0.180 mumoles NH3/min/mg protein. By addition of cycloheximide it was shown that ammonia influences the synthesis of this enzyme. Enzyme induction by the substrate could be excluded. Even under the conditions of highest repression a specific activity of urease of 0.180 mumoles NH3/min/mg protein was measured. Possible causes of this constitutive enzyme level are discussed.     

Physico-Chemical Characterization of Watermelon Urease upon Immobilization in Alginate Beads

Watermelon (Citrullus vulgaris) urease was immobilized in 3.5% alginate leading to 72% immobilization. There was no leaching of the enzyme over a period of 15 days at 4°C. It continued to hydrolyse urea at a faster rate upto 90 min of incubation. The immobilized urease exhibited a shift of apparent pH optimum by one unit towards acidic side (from pH 8.0 to 7.0). The K_m was found to be 13.3 mM; 1.17 times higher than the soluble enzyme (11.4 mM). The beads were fairly stable upto 50°C and exhibited activity even at ?10°C. The enzyme was significantly activated by ME and it exhibited two peaks of activation; one at lower concentration and another at higher concentration. Time-dependent ureolysis in presence of ME progressed at a much elevated rate. Unlike soluble enzyme, which was inhibited at 200 mM urea, the immobilized enzyme was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2000 mM urea. Moreover, the inhibition caused by high urea concentration was partially abolished by ME. The significance of the observations is discussed.     

Urea metabolism in isolated perfused lungs of the laboratory rat and of a desert rodent, Notomys alexis

1. Using the isolated perfused lung preparation we have demonstrated a low-activity ureolytic enzyme present in rodent lung tissue. The enzyme shares four characteristic features with jack bean urease (EC 3.5.1.5). 2. Ureolytic activity was inhibited by fluoride ions and methionine hydroxamic acid; using the latter inhibitor, the I50 value and maximum inhibition were similar to those reported for jack bean urease. The apparent Km for rat lung urease was similar to the plasma urea level. 3. The low level of urease activity in the rat lung and in that of Notomys alexis, a desert rodent, suggests that the enzyme is not involved in urea excretion, rather that pulmonary ammonia production may influence fluid balance at the alveolus.     

Lichen substrates and urease production and secretion: A physiological approach using four Antarctic species

Ramalina terebrata, C. regalis and S. alpinum produce urease in response to urea exogenously supplied. Enzyme activity is abolished by including 300 μM cycloheximide in the incubation media. Urease is affected by feed-back inhibition when ammonia is accumulated in the thalli. Lecania brialmontii has a constitutive urease which is strongly inhibited by an excess of urea. The enzyme is secreted to the media as a function of the soil in which lichens survive.     

脲对棒状链霉菌棒酸合成的影响

【目的】棒酸(Clavulanic acid)是棒状链霉菌(Streptomyces clavuligerus)产生的β-内酰胺酶抑制剂,其合成过程中产生副产物脲,旨在探讨脲对棒酸合成的影响。【方法】通过发酵过程中脲和铵盐添加实验、阻断脲酶活性以及pH梯度实验研究脲对棒酸合成影响。【结果】脲添加实验结果表明:低浓度脲降低棒酸产量,当添加脲浓度达到20 mmol/L时,完全抑制棒酸合成。由于脲酶可以把脲水解为铵离子,导致铵离子浓度及pH提高,因此,通过阻断棒状链霉菌脲酶活性,可以更准确地反映脲对棒酸合成的影响。结果发现,脲酶敲除株发酵液中脲大量积累,浓度高达10 mmol/L,但棒酸产量没有明显降低,说明在该浓度下脲自身并不能抑制棒酸合成。添加脲降低野生菌棒酸产量,可能是脲被水解为铵离子或其引起的pH变化所致。而棒酸发酵液添加铵盐的结果显示铵离子对棒酸产量没有抑制作用;另外,pH梯度实验证实不同pH对棒酸产量影响较大。【结论】排除了脲和铵离子对棒酸合成的抑制作用,证实了脲酶水解脲导致pH提高是脲添加导致野生菌棒酸产量降低的真正原因,为进一步阐明棒酸合成调控机制提供了根据。     

Urease from leaves of Glycine max and Zea mays

Extracts of Glycine max and Zea mays leaves catalysed the release of ¹⁴CO₂ from [¹⁴C] urea with multiple pH maxima (5.5 and 9.0 for G. max; 5.5, 7.5 and 8.8 for Z. mays). Evidence was obtained that the principal activities, at pH 5.5 and 8.8–9.0 catalysed the same reaction stoichiometry as did urease purified from jackbean seeds (EC 3.5.1.5). The urease activities with these pH optima were not resolved by ammonium sulphate fractionation, DEAE-cellulose chromatography, or gel filtration chromatography. Many structural analogues of urea inhibited leaf urease, the most effective being amino acid hydroxamates, hydroxyurea and selenourea. Allantoic acid and ureidoglycolate are probably not alternative substrates because they showed at most only weak competitive inhibition with respect to radioactive urea.     

Characterization of urease from the phototrophic bacteriumRhodobacter capsulatus E1F1

Rhodobacter capsulatus E1F1 showed high cytosolic urease activity when growing on urea, purines, and purine metabolites as nitrogen source. Molecular mass ofR. capsulatus enzyme is similar to that of other bacteria and greatly differs from that of jack bean. Kinetic parameters of partially purifiedR. capsulatus enzyme resemble those described in other bacterial ureases. The activity was inhibited by metal-chelating agents and by mercurials. Urease fromR. capsulatus E1F1 was negligible in nitrogen-starved cells or in cells cultured with nitrate, ammonium, or amino acids. Moreover, ammonium inhibited both the urea uptake and the urease activity expression inR. capsulatus cells.     

Inhibition of urease by Ni ions: Analysis of reaction progress curves

The inhibition of jack bean urease by Ni²⁺ ions was studied in 20 mM HEPES buffer pH 7.0. The inhibition was observed in two systems which differed in the order in which the components of the reaction mixture were mixed. In the first (unincubated), the reaction was initiated by adding urease to the mixture of urea and Ni²⁺ ions, and in the second (incubated), by adding urea to the mixture of urease incubated with Ni²⁺ ions prior to the reaction. It was shown that Ni²⁺ ions are a competitive slow-binding inhibitor of urease. In the first system the inhibition constants are K_i=0.042 mM and K_i^*=0.0028 mM, and in the second system K_i^*=0.0024 mM. The inhibition was found to involve the rapid formation of a urease-Ni²⁺complex followed by its relatively slow, reversible isomerization, with forward and reverse rate constants of 0.64 and 0.045 min⁻¹, respectively.     

Nickel availability and urease expression in Proteus mirabilis

Cells of Proteus mirabilis, previously grown in nutrient broth (NB), exhibited an increase in urease activity during subsequent incubation in mineral medium even when protein biosynthesis was inhibited. During growth in NB, degradation of amino acids obviously led to the formation of nickel-complexing metabolites, and nickel ions were therefore inavailable for maximal expression of enzymatically active urease; this inhibition of urcase biosynthesis was overcome by the addition of nickel to the growth medium, and also by added glucose. Experiments concerning the incorporation of radioactive nickel into urease finally indicated that the observed increase in urease activity was caused by posttranslational insertion of nickel into preformed apourease.     

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.     

Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze the hydrolysis of urea. Here, we describe the steady-state and pre-steady-state kinetics of urease inhibition by fluoride. Urease is slowly inhibited by fluoride in both the presence and absence of substrate. Steady-state rate studies yield parallel double-reciprocal plots; however, we show that fluoride interaction with urease is not compatible with classical uncompetitive inhibition. Rather, we propose that fluoride binds to an enzyme state (E) that is in equilibrium with resting enzyme (E) and produced during catalysis. Fluoride binding rates are directly proportional to inhibitor concentration. Substrate reduces both the rate of fluoride binding to urease and the rate of fluoride dissociation from the complex, consistent with urea binding to E and E.F in addition to E. Fluoride inhibition is pH-dependent due to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual inhibitor. We assess the kinetic results in terms of the known protein crystal structure and evaluate possible molecular interpretations for the structure of the E state, the site of fluoride binding, and the factors associated with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may need to be reinterpreted in terms of fluoride interaction with the corresponding E states.     

新型磷酰胺类脲酶抑制剂对不同质地土壤尿素转化的影响

施用脲酶抑制剂是降低尿素水解、减少氨气挥发损失、提高作物氮(N)肥利用率的重要途径之一.采用室内恒温、恒湿模拟试验方法,在25 ℃黑暗条件下培养,研究新型磷酰胺类脲酶抑制剂N-丙基磷酰三胺(NPPT)的脲酶抑制效果,比较其与N-丁基磷酰三胺(NBPT)在不同尿素用量条件下不同质地土壤中对脲酶的抑制差异.结果表明: 在壤土和黏土中,尿素作用时间≤9 d,添加抑制剂可以将尿素水解时间延长3 d以上.砂土中,尿素分解过程相对缓慢,添加抑制剂显著降低土壤脲酶活性,抑制NH₄⁺-N生成.在培养期间,不同尿素用量条件下,脲酶抑制剂在不同质地土壤中的抑制效果表现为高施N量优于低施N量.培养第6天,在尿素用量250 mg N·kg^-1条件下,NBPT和NPPT在砂土中脲酶抑制率分别为56.3%和53.0%,在壤土中分别为0.04%和0.3%,在黏土中分别为4.1%和6.2%;尿素用量500 mg N·kg^-1,NBPT和NPPT在砂土中脲酶抑制率分别为59.4%和65.8%,在壤土中分别为14.5%和15.1%,在黏土中分别为49.1%和48.1%.不同质地土壤中脲酶抑制效果表现为砂土>黏土>壤土.不同抑制剂处理在培养期间土壤NH₄⁺-N含量呈现先上升后下降的趋势,而NO₃^--N含量和表观硝化率均呈现逐渐上升的趋势.与单施尿素处理相比,添加脲酶抑制剂NBPT和NPPT显著增加土壤中的残留尿素态N,降低NH₄⁺-N生成.新型脲酶抑制剂NPPT在不同质地土壤中的抑制效果与NBPT相似,是一款有效的脲酶抑制剂.     

Inhibition of endogenous urease activity by NBPT application reveals differential N metabolism responses to ammonium or nitrate nutrition in pea plants: a physiological study

Background and aims

Urea is the predominant form of N applied as fertilizer to crops, but it is also a significant N metabolite of plants themselves. As such, an understanding of urea metabolism in plants may contribute significantly to subsequent N fertilizer management. It currently appears that arginase is the only plant enzyme that can generate urea in vivo. The aim of this work was, therefore, to gain a more in-depth understanding of the significance of the inhibition of endogenous urease activity and its role in N metabolism depending on the N source supplied.

Methods

Pea (Pisum sativum cv. Snap-pea) plants were grown with either ammonium or nitrate as the sole N source in the presence or absence of the urease inhibitor NBPT.

Results

When supplied, NBPT is absorbed by plants and translocated from the roots to the leaves, where it reduces endogenous urease activity. Different N metabolic responses in terms of N-assimilatory enzymes and N-containing compounds indicate a different degree of arginine catabolism activation in ammonium- and nitrate-fed plants.

Conclusions

The arginine catabolism is more highly activated in ammonium-fed plants than in nitrate-fed plants, probably due to the higher turnover of substrates by enzymes playing a key role in N recycling and remobilization during catabolism and in early flowering and senescence processes, usually observed under ammonium nutrition.     

Urease from Arthrobacter oxydans,a nickel-containing enzyme

In Arthrobacter oxydans, Klebsiella aerogenes and Sporosarcina ureae, growth with urea as a nitrogen source turned out to be more sensitive to inhibition by EDTA than that with ammonia. The inhibition was overcome by added nickel chloride, but not by other divalent metal ions tested. In A. oxydans the uptake of ⁶³Ni was paralleled by an increase in urease (urea amidohydrolase, EC 3.5.1.5) activity under certain conditions. Following growth with radioactive nickel, urease from this strain was enriched by heat treatment and acetone fractionation. Copurification of ⁶³Ni and urease was observed during subsequent Sephadex gel chromatography. Almost the entire labelling was detected together with the purified enzyme after focusing on polyacrylamide gel. The relative molecular mass of the purified urease was estimated to be 242,000. The pH optimum was 7.6, the K _m-value 12.5 mmol/l and the temperature optimum 40°C; heat stability was observed up to 65°C. In presence of 10 mmol/l EDTA the protein-nickel binding remained intact at pH 7; at pH 5 and below, nickel was irreversibly removed with concommitant loss of enzyme activity. The results demonstrated that nickel ions are required for active urease formation in the bacterial strains studied, and that urease from A. oxydans is a nickel-containing enzyme.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

A Possible Role for Urease as a Storage Protein in Aspergillus tamarii

A marked decrease in mycelial urease activity during the endogenousphase of undifferentiated Aspergillus tamariicultures was foundto be independent of preparative procedures but related to thedepletion of external nutrients. The enzyme, which was synthesizedduring the active growth stage, was produced in similar quantitieswith ammonium or urea as sole nitrogen source and at its peakrepresented c. 8·5 per cent of the total soluble proteinpool of the mycelium. It was found to show maximum activityat pH 8·20–8·65 when measured in cell-free,phosphate-buffered extracts. Isolation of urease from differentstages of the endogenous phase by affinity chromatography hasshown that the observed decrease in activity was due to breakdownof the enzyme protein in mature cultures, followed by the progressivedeactivation of residual enzyme during the autolytic stage.Since selective inhibition of 80–90 per cent of activityby acetohydroxamic acid in media containing urea as the onlynitrogen source or total repression of urease synthesis by L-histidinein ammonium-grown cultures did not interfere with normal growth,it was concluded that in A. tamarii urease fulfils the functionof a storage protein with a measure of catalytic activity. Aspergillus tamarii, urease, storage protein, nitrogen metabolism     

Irreversible Inhibition of Jack Bean Urease by Pyrocatechol

Pyrocatechol was studied as an inhibitor of jack bean urease in 20?mM phosphate buffer, pH 7.0, 25°C. The inhibition was monitored by an incubation procedure in the absence of substrate and reaction progress studies in the presence of substrate. It was found that pyrocatechol acted as a time- and concentration dependent irreversible inactivator of urease. The dependence of the residual activity of urease on the incubation time showed that the rate of inhibition increased with time until there was total loss of enzyme activity. The inactivation process followed a non-pseudo-first order reaction. The obtained reaction progress curves were found to be time-dependent. The plots showed that the rate of the enzyme reaction in the final stages reached zero. From protection experiments it appeared that thiol-compounds such as l-cysteine, 2-mercaptoethanol and dithiothreitol prevented urease from pyrocatechol inactivation as well as the substrate, urea, and the competitive inhibitor boric acid. These results proved that the urease active site was involved in the pyrocatechol inactivation.     

Irreversible inhibition of jack bean urease by pyrocatechol

Pyrocatechol was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 25 degrees C. The inhibition was monitored by an incubation procedure in the absence of substrate and reaction progress studies in the presence of substrate. It was found that pyrocatechol acted as a time- and concentration dependent irreversible inactivator of urease. The dependence of the residual activity of urease on the incubation time showed that the rate of inhibition increased with time until there was total loss of enzyme activity. The inactivation process followed a non-pseudo-first order reaction. The obtained reaction progress curves were found to be time-dependent. The plots showed that the rate of the enzyme reaction in the final stages reached zero. From protection experiments it appeared that thiol-compounds such as L-cysteine, 2-mercaptoethanol and dithiothreitol prevented urease from pyrocatechol inactivation as well as the substrate, urea, and the competitive inhibitor boric acid. These results proved that the urease active site was involved in the pyrocatechol inactivation.     

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.     

Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction.

Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea. The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate. Urease activity was readily detectable in M. tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium. The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria. The nucleotide sequence of the M. tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G.     

Urease immobilized on an aminated polysulphone membrane – inhibition by boric acid

An enzymatic membrane for application in the processes of decomposition and removal of urea from aqueous solutions was prepared: jack bean urease was immobilized on an aminated polysulphone membrane by adsorption. The inhibition of the system by boric acid was studied using procedures based on the MICHAELIS-MENTEN integrated equation (non-linear regression, and the linear transformations of WALKER and SCHMIDT, JENNINGS and NIEMANN, and BOOMAN and NIEMANN). The reaction was carried out in a 100 mM phosphate buffer of pH 7.0, containing 2 mM EDTA, obtained by neutralization of orthophosphoric acid with NaOH, at an initial urea concentration of 10 mM, and a temperature of 25 °C. The reaction was initiated by the addition of the enzyme to the urea solution, and was monitored by removing samples of the reaction mixture for NH₃ determinations by the phenol-hypochlorite method until the urea was exhausted. The results were compared with those obtained earlier under the same reaction conditions for free urease and urease covalently immobilized on chitosan. The inhibition was found to be competitive, similar to that of the free enzyme and urease immobilized on chitosan, with inhibition constants K_i equal to 0.36, 0.19 and 0.60 mM. The results show that adsorption of the enzyme on a polysulphone membrane changed the enzyme to a lesser degree than covalent immobilization of the enzyme on a chitosan membrane.