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)

Urease (urea amidohydrolase; EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbamate. The latter compound spontaneously decomposes to yield another molecule of ammonia and carbonic acid. The urease phenotype is widely distributed across the bacterial kingdom, and the gene clusters encoding this enzyme have been cloned from numerous bacterial species. The complete nucleotide sequence, ranging from 5.15 to 6.45 kb, has been determined for five species including Bacillus sp. strain TB-90, Klebsiella aerogenes, Proteus mirabilis, Helicobacter pylori, and Yersinia enterocolitica. Sequences for selected genes have been determined for at least 10 other bacterial species and the jack bean enzyme. Urease synthesis can be nitrogen regulated, urea inducible, or constitutive. The crystal structure of the K. aerogenes enzyme has been determined. When combined with chemical modification studies, biophysical and spectroscopic analyses, site-directed mutagenesis results, and kinetic inhibition experiments, the structure provides important insight into the mechanism of catalysis. Synthesis of active enzyme requires incorporation of both carbon dioxide and nickel ions into the protein. Accessory genes have been shown to be required for activation of urease apoprotein, and roles for the accessory proteins in metallocenter assembly have been proposed. Urease is central to the virulence of P. mirabilis and H. pylori. Urea hydrolysis by P. mirabilis in the urinary tract leads directly to urolithiasis (stone formation) and contributes to the development of acute pyelonephritis. The urease of H. pylori is necessary for colonization of the gastric mucosa in experimental animal models of gastritis and serves as the major antigen and diagnostic marker for gastritis and peptic ulcer disease in humans. In addition, the urease of Y. enterocolitica has been implicated as an arthritogenic factor in the development of infection-induced reactive arthritis. The significant progress in our understanding of the molecular biology of microbial ureases is reviewed.
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PMID:Molecular biology of microbial ureases. 756 14

In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.
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PMID:Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation. 772 85

Assembly of protein metallocenters is not well understood. Urease offers a tractable system for examination of this process. Formation of the urease metallocenter in vivo is known to require four accessory proteins: UreD, postulated to be a urease-specific molecular chaperone; UreE, a nickel(II)-binding protein; and UreF and UreG, of unknown function. Activation of purified Klebsiella aerogenes urease apoprotein was accomplished in vitro by providing carbon dioxide (half-maximal activation at approximately 0.2 percent carbon dioxide) in addition to nickel ion. Activation coincided with carbon dioxide incorporation into urease in a pH-dependent reaction (pKa > or = 9, where Ka is the acid constant). The concentration of carbon dioxide also affected the amount of activation of UreD-urease apoprotein complexes. These results suggest that carbon dioxide binding to urease apoprotein generates a ligand that facilitates productive nickel binding.
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PMID:Requirement of carbon dioxide for in vitro assembly of the urease nickel metallocenter. 785 93

The formation of active urease in Klebsiella aerogenes requires the presence of three structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the metallocenter in this nickel-containing enzyme. Slow and partial activation of urease apoprotein was observed after addition of nickel ion to extracts of Escherichia coli cells bearing a plasmid containing the K. aerogenes urease gene cluster or derivatives of this plasmid with deletions in ureE, ureF, or ureG. In contrast, extracts of cells containing a ureD deletion derivative failed to generate active urease, thus highlighting a key role for UreD in the metallocenter assembly process. Site-directed mutagenesis methods were used to overexpress ureD in the presence of the other urease genes, and the UreD protein was found to copurify with urease. A molecule of native urease apoprotein is capable of binding 0, 1, 2, or 3 molecules of UreD, consistent with a trimeric structure of urease catalytic units. The UreD-urease apoprotein complexes are competent for activation by nickel, with the level of activity obtained being directly related to the number of UreD molecules bound per urease molecule. Activation of the UreD-urease complexes is rapid and accompanied by UreD dissociation. We propose that UreD is a chaperone protein which stabilizes a urease apoprotein conformation that is competent for nickel incorporation.
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PMID:In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly. 790 61

Four microbial enzymes are known to require nickel: hydrogenase, methyl coenzyme M reductase, carbon monoxide dehydrogenase, and urease. Recent biochemical and molecular biological experiments have provided clear evidence for the existence of multiple auxiliary genes that facilitate nickel incorporation into urease and hydrogenase. Similarly, accessory factors are also likely to be required for the other two enzymes. One of the urease-related genes (ureE) encodes a cytoplasmic protein that has been purified and shown to bind nickel reversibly. We propose that the UreE protein serves as a nickel donor to urease apoprotein. A second urease-related auxiliary gene (ureG) possesses a sequence motif that is found in ATP- and GTP-binding proteins. We have shown that nickel incorporation into urease requires energy and speculate that the UreG protein may serve as an energy transducer, coupling the energy of NTP hydrolysis to metallocenter incorporation. The UreG protein is related in sequence to HypB, a protein that has been proposed to function in nickel processing in hydrogenases. Hence, the mechanisms for metallocenter biosynthesis in these two dissimilar enzymes may have evolved from a common nickel incorporation system.
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PMID:Nickel enzymes in microbes. 802 91

The Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174, 4324-4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic beta-strand and exhibits unusually tight binding to phenyl-Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (M(r) = 35,000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 +/- 1.3 microM) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X-ray absorption and variable-temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo-octahedral geometry with 3-5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X-ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.
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PMID:Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly. 831 89

Klebsiella aerogenes urease in a Ni-containing enzyme (two Ni per alpha beta gamma unit) that is purified as an apoprotein from cells grown in Ni-free medium. Partial activation of urease and UreD-urease apoproteins is achieved in vitro by incubation in the presence of Ni(II) and CO2, whereas incubation of these proteins with Ni alone leads to the formation of inactive species [Park, I.-S., & Hausinger, R. P. (1995) Science 267, 1156-1158]. Here we determined the kinetics of these inhibitory reactions and demonstrated the presence of two Ni ions per alpha beta gamma unit in the inactive proteins. Although metal-substituted urease has never been purified from Ni-deprived cell, several other metal ions were shown to bind to the urease apoproteins. Divalent Zn, C, Co, and Mn all inhibited Ni- and Co2-promoted urease activation at concentrations below that of Ni, whereas Mg and Ca ions did not inhibit this process. Ni-inhibited species recovered their ability to be partially activated after EDTA treatment. In contrast, samples that were exposed to Co or Cu ions were irreversibly inactivated, and EDTA treatment of Zn- or Mn-inhibited samples led to reduced levels of activation competence. Mn-substituted urease, generated from urease apoprotein samples in a Mn- and Co2-dependent manner, was shown to be active, whereas other metal-substituted forms if urease lacked activity. The Mn-protein possessed only 2% of the activity of Ni-activated apoprotein [ approximately 8.0 vs approximately 400 mumol min-1 (mg protein)-1], but its KM value was only moderately altered from that of the native enzyme (3.86 +/- 0.15 mM vs 0.2 mM). Unlike the Ni-containing enzyme, Mn-urease was inhibited by EDTA. Given the evidence that urease apoprotein binds numerous metal ions, we speculate on possible roles for the UreD, UreF, and UreG accessory proteins in urease activation.
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PMID:Metal ion interaction with urease and UreD-urease apoproteins. 861 23

In vivo assembly of the Klebsiella aerogenes urease nickel metallocenter requires the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. Prior studies had shown that urease apoprotein exists in an uncomplexed form as well as in a series of UreD-urease (I.-S. Park, M.B. Carr, and R.P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S. Park and R.P. Hausinger, J. Bacteriol. 177:1947-1951, 1995) apoprotein complexes. This study demonstrates the existence of a distinct series of complexes consisting of UreD, UreF, and urease apoprotein. These novel complexes exhibited activation properties that were distinct from urease and UreD-urease apoprotein complexes. Unlike the previously described species, the UreD-UreF-urease apoprotein complexes were resistant to inactivation by NiCl2. The bicarbonate concentration dependence for UreD-UreF-urease apoenzyme activation was significantly decreased compared with that of the urease and UreD-urease apoproteins. Western blot (immunoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF. We propose that the binding of UreF modulates the UreD-urease apoprotein activation properties by excluding nickel ions from binding to the active site until after formation of the carbamylated lysine metallocenter ligand.
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PMID:Purification and activation properties of UreD-UreF-urease apoprotein complexes. 880 30

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

Urease possesses a dinuclear nickel active site with the metals bridged by a carbamylated lysine residue. In vitro activation of apoprotein (Apo) is achieved by incubation with Ni(II) and bicarbonate as a source of CO2. Analogues of CO2 and bicarbonate were examined for their effects on the Apo activation process. While SO2 had little effect, CS2 was shown to inhibit Apo activation via its ability to substitute for CO2 to yield an inactive dithiocarbamate-containing protein. Sulfur-to-Ni charge-transfer transitions arising from this species yielded an electronic absorption band at 324 nm with a shoulder at 382 nm. Borate, sulfate, phosphate, and molybdate had essentially no effect on Apo activation and did not substitute for bicarbonate, while treatment of Apo with Ni(II) plus vanadate led to the production of active urease containing two Ni and one V per active site. Vanadate-dependent activation of Apo resembled the normal activation process in terms of concentration of anion required, optimal pH, and incubation time needed. Furthermore, the UV-visible spectrum, maximal specific activity [386 +/- 26 U.(mg of protein)-1], Km (1.83 +/- 0.20 mM urea), and pH dependence for the vanadate-containing urease were essentially identical to properties observed for bicarbonate-activated enzyme. Vanadate-activated Apo is proposed to possess a vanadylated lysine that bridges the two Ni ions comprising its metallocenter.
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PMID:Substitution of the urease active site carbamate by dithiocarbamate and vanadate. 939 39


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