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Urease has an EC number: 184.108.40.206. It is a metalloenzyme, which is involved as a catalyst, in the hydrolysis of urea to yield ammonia and carbon dioxide (Dixon et al., 1975). Urease can be found in plants, fungi and bacteria ([Mobley and Hausinger, 1989],Â [Polacco and Havir, 1979],Â [Hirayama et al., 2000]Â andÂ [Cox et al., 2000]). Acetamide, formamide,Â N-methylurea, semicarbazide, and hydroxyurea are also hydrolysed by urease (Dixon et al., 1980a). In case of urea hydrolysis, plant and microbial ureases have KmÂ values ranging from 0.1 to >100Â mM urea; jack bean urease (JBU) has a KmÂ value of 2.9Â mM (Mobley and Hausinger, 1989). Externally applied as fertilizer, urea becomes accessible to plants only through urease activity (Witte et al., 2002). Since urea is one of the world's most widely used nitrogen fertilizers, its enzymatic hydrolysis is a process of great agricultural importance. In addition, urease is known to be a major cause of pathologies induced byÂ Helicobacter pylori, as it allows the bacteria to survive at the low pH of stomach during colonization and therefore plays an vital role in the pathogenesis of gastric and peptic ulcers, which in some cases may lead to cancer (Mobley et al., 1995). In agriculture, high urease activity causes significant environmental and economic problems by releasing abnormally large amounts of ammonia into the atmosphere during urea fertilization ([Mobley et al., 1995],Â [Mobley and Hausinger, 1989]Â andÂ [Sirko and Brodzik, 2000]). Besides the importance of urease in making nitrogen available to plants and the impact of microbial ureases in agriculture and medicine, the structure and catalytic mechanism of this enzyme are of interest because of its large enhancement (1014-fold) of the rate of urea hydrolysis and the presence of active-site nickel, which is unique among hydrolytic enzymes (Estiu et al., 2006).
Biochemically, the best-characterized plant urease is that from jack bean (Canavalia ensiformis) ([Zerner, 1991],Â [Sheridan et al., 2002],Â [Follmer et al., 2004a]Â and[Krajewska and Ciurli, 2005]). JBU was the first enzyme to be crystallized, and it played an important historical role as proof of the proteinaceous nature of enzymes. SumnerÂ´s work, ''Isolation and crystallization of the enzyme urease" (Sumner, 1926, Nobel Prize for Chemistry, 1946), represents a fundamental contribution to modern enzymology. JBU is also the first nickel-containing enzyme to be described (Dixon et al., 1975), and it is the only nickel-containing metalloenzyme identified so far in plants (Polacco and Holland, 1993). Unfortunately, neither the three-dimensional structure of JBU nor that of any other plant urease has been determined. The great heterogeneity, insolubility, and high polydispersity of JBU in solution ([Fishbein et al., 1969],Â [Fishbein and Nagarajan, 1971],Â [Fishbein, 1969]Â andÂ [Follmer et al., 2004a]) may be related to the lack of success in determining its structure by X-ray crystallography ([Jabri et al., 1992]Â andÂ [Sheridan et al., 2002]). Moreover, the fact that JBU is not the only urease expressed inÂ C. ensiformisÂ (Pires-Alves et al., 2003) raises the possibility that contamination by urease isoforms during JBU purification impedes the production of adequate JBU crystals. Therefore, most of our knowledge about the molecular mechanism of ureolytic catalysis by plant ureases is based on the 3-D structures of bacterial ureases.
STRUCTURAL CHARACTERISTICS OF UREASES
JBU exists as homotrimers that can associate to form hexamers of identicalÂ 90Â kDa subunits containing two nickel ions per subunit ([Dixon et al., 1975],Â [Dixon et al., 1980]Â andÂ [Takishima et al., 1988]). Bacterial ureases are similar to JBU in that they are either trimers or hexamers of complex subunits (Mobley et al., 1995). Fungal and plant ureases such as jack bean and soybean are found to be homo-oligomeric proteins ofÂ aroundÂ 90Â kDa subunits, whereas bacterial ureases are found to be multimers of 2 or 3 subunit complexes (Mobley et al., 1995). The A, B and C chains ofÂ Klebsiella aerogenes,Â Bacillus pasteuriiÂ and most other bacterial ureases are co-linear with the single chain of fungal and plant ureases.Â H. pyloriÂ urease has two subunits: a small polypeptide chain (A chain - 238 residues) homologous to the N-terminal region of JBU and a larger chain (B chain - 569 amino acids) containing the catalytic site. Although only bacterial ureases such asÂ K. aerogenesÂ (Jabri et al., 1995),Â B. pasteuriiÂ (Benini et al., 1999) andÂ H. pyloriÂ urease (Ha et al., 2001), as well as several mutants and inhibited systems have their tertiary structures successfully solved by X-ray crystallography, there are significant amino acid sequence similarities among all known ureases (Mobley et al., 1995). Comparing the structures of microbial ureases we can establish extensive similarity of both the secondary and tertiary structures despite differences in the number of chains.
Â Similarity of the structures of bacterial ureases [Klebsiella aerogenesÂ urease (KAU) (PDB code: 1FWJ); Bacillus pasteuriiÂ urease (BPU) (PDB code 2UBP);Â Helicobacter pyloriÂ urease (HPU) (PDB code 1E9Z)].
Expression of the urease operon is sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions, which is controlled by the organism Streptococcus thermophilus (Arioli, Monnet, 2007). The expression of JBU and JBURE-II genes is induced in seedlings and in leaves treated with abscisic acid, a phytohormone involved in seed maturation and wound reponse, some of the organisms involved in this prosess are Canavalia ensiformis, Morus alba, Zea mays, Glycine max. (Davis, H.M.; Shih, L.M., 1984). Soybean contains an ubiquitous urease (encoded by Eu4) that is synthesized in all tissues, as well as an embryo-specific urease (encoded by the gene Eu1) that is confined to the developing embryo and is retained in mature seeds where its activity is roughly 1000fold higher than that of ubiquitous urease (Follmer, 2007) which is governed by the organism Gossypium hirsutum.
Urease is localized in the Cytoplasm of Methylophilus methylotrophus, Cytosol of Rhodobacter capsulatus or intracellular region of Rhizopus oryzae. (Geeweely, 2006) (Pineda, 1993)
METHODS OF ISOLATION & EXTRACTION
Using a mortar and pestle, 3 g ofÂ freshÂ plant material is homogenised with 9 ml of extraction buffer (50 mM sodium phosphate buffer (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1.5% (w/v) polyvinylpolypyrollidone (PVPP), 10 mM dithiothreithol (DTT) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)). DTT and PMSF have to be added shortly before the extraction.Â The plant material should be fresh, since urease is may be partially inactivated in the thawing process. The homogenised tissue is transferred to a 15-ml centrifuge tube and the total extract volume is determined (for example, by comparing the filling height with to a scale on a similar tube). The tube is centrifuged at 10000 g for 10 minutes to remove coarse debris, the supernatant transferred into a new tube and centrifuged at 40000 g for 20 minutes. The clarified supernatant is stored on ice. In order to be able to use theÂ phenol hypochloride reagentsÂ for urease quantification, low molecular substances that interfere with the assay chemistry usually have to be removed. The DTT added during urease extraction is not compatible with the phenol hypochloride chemistry and plant extracts might contain further low molecular substances that interfere.Â A 5-ml High Trap G25 desalting column (Pharmacia) is used to eliminate interfering low molecular weight substances. The column is equilibrated with four column volumes of gel filtration buffer (25 mM sodium phosphate buffer, pH 7.5; 25 mM NaCl; 0.5 mM EDTA) at the beginning and between individual runs. A flow rate of 6 ml/min is applied with a peristaltic pump. For sample processing, the pump is disconnected, a sample of 1.5 ml is applied with a 2.0 ml syringe, the pump is reconnected, and 2.0 ml are immediately recovered from the column outlet in a 2 ml microfuge tube. If you determine the urease activity per unit fresh weight on an extract prepared in this way, you may consider the dilution caused by the gel filtration step with a factor 1.35 (2.0 ml / 1.5 ml). You can also test your real dilution by measuring the total protein content before and after the gel filtration step. In my experience the real dilution is usually between 1.3 and 1.5. (Claus-Peter WitteÂ andÂ Nieves Medina-Escobar,Â 2001)
Optimum pH of Jack bean Urease is at pH 7.4, whereasÂ there are two optimum pH for H. pyloriÂ urease of 4.6 and 8.2. KmÂ for H. Pylori was found to be 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tis Â· HCl at pH 8.2. On the other hand, jack bean urease has aÂ KmÂ of 1.3 mM in TrisÂ·HCl. (Stephaine D Cesareo, 2002)
Purified enzyme from rumen bacterial fraction of bovine rumen content has an optimum pH at pH 8.0. The molecular weight has been found to be 120000-130000. The Km for was also found to be 8.3 X 10(-4) M+/-1.7 X 10(-4) M. (Mahadevan S, Sauer FD, Erfle JD, 1977).
Urea can be utilized as a nitrogen source via a urease-dependent pathway, the ureolysis can protect the organism against environmental acidification at physiologically relevant pH values. Therefor, urease can confer to Actinomyces naeslundii critical selective advantages over nonureolytic organisms in dental plaque. (Morou-Bermudez E,Â Burne RA, 1999).
Cotton seed urease displays low ureolytic activity but exhibits potent antifungal properties at sub-micromolar concentrations against different phytopathogenic fungi. The antifungal effect of cotton urease persists after treatment with an irreversible inhibitor of its enzyme activity. The data suggest an important role of the protein in plant defense. (Menegassi A,Â Wassermann GE, 2008).
As a result of the inhibitors, the cysteines flaps are protected from reacting further with the disulphides, thus resulting in the formation of closed flaps. Inhibition for urease occurs in both noncompetitive as well as concentration dependent manner. (Amtul Z,Â Follmer C, 2007) (Krajewska B,Â Zaborska W, 2007)
Urease is much more stable in the presence of nickel located inside the enzyme. Urease from a gram positive coccoid isolate prototype was obtained from an organism, which contributes to a large part of the total cell proteins. Urease using gel electrophoresis was purified to 138-fold to apparent homogeneity. The urease was found to be unstable during purification, when nickel was absent, nickel is generally found in a metallocenter located in other microbial ureases. In the presence of nickel sulfate during growth as well as in buffers during sonication and purification, the urease was found to be having stability during the purification process at room temperature. In the native state, urease has a molecular weight of 260 kDa and consists of 3 subunits of 65 kDa and 3 subunits of 21 kDa. In the purified state, urease was found to relatively stable in acidic conditions and its activity was not degraded after when it was incubated for 30 mins at pH 1.3. After measuring the K(m)s for urease for both whole cells as well as purified enzyme, values were found to be 0.56 and 1.7 mM, respectively, which shows that some cell wall component(s) alters, enzymes affinity for urea. V(max) was calculated for urea hydrolysis and V(max) for whole cells was 8.1 mol/min/mg and for purified enzyme was 8.1 and 1,120 mol/min/mg of protein. (Lee SG,Â Calhoun DH, 1997)
RELEVANACE OF UREASE IN FOOD
Acid urease derived from Lactobacillus fermentum, however, exhibits its highest rate of urea decomposition at acid pH conditions and can therefore be utilized for effective reduction of urea in wine (Kakimoto et al., 1989). The effectiveness of this enzyme in wine depends strongly on the type wine in which it is used and the conditions. Reports indicate the presence of inhibitors of urease in wines such as fluoride, malate, ethanol, and phenolic compounds (Trioli and Ough, 1989; Famuyiwa et al., 1991; Kodama et al., 1991). Acid urease has been affirmed as Generally Recognized As Safe by the Food and Drug Administration of the US for use in wine, including sake (1992). However, even when the urea content is below detectable levels, complete prevention of EC formation could not be achieved by urea degradation alone. There are other precursors such as citrullin and other carbamyl compounds (Ough et al., 1988b; Tegmo-Larsson and Henick-Kling, 1990; Stevens and Ough, 1993).