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)

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.
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PMID:Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. 131 13

Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.
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PMID:Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. 132 Jun 7

The DNA sequence of the urea amidolyase (DUR1,2) gene from S. cerevisiae has been determined. The polypeptide structure deduced from the DNA sequence contains 1,835 amino acid residues and possesses a calculated weight of 201,665 daltons which favorably correlates with that predicted from compositional analysis of purified protein (1,881 amino acid residues and a molecular weight of 203,900). The C-terminal 57 residues of the polypeptide exhibit significant homology with similarly situated sequences found in five other biotin carboxylases whose primary structures have been determined or deduced from protein and DNA sequence data, respectively. Major S1 nuclease protection fragments derived from DUR1,2 RNA-DNA hybrids exhibit apparent termini at positions -140 and -141 upstream of the coding region. The termini of minor protection fragments also occur at eleven other positions as well.
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PMID:The urea amidolyase (DUR1,2) gene of Saccharomyces cerevisiae. 180 34

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.
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PMID:Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus. 184 42

Helicobacter pylori (formerly Campylobacter pylori) is the causative agent of gastritis in man. Helicobacter pylori cells contain a large amount of an extremely active urease (E.C.3.5.1.5). This enzyme is suspected to be a virulence factor since the ammonium ion produced from urea may be responsible for tissue injury and/or survival of H. pylori in the gastric environment. Helicobacter pylori urease, native relative molecular mass approximately 600,000, was purified by agarose gel filtration and ion exchange chromatography. DEAE-purified urease is highly active and has a Km of 0.48 mM for urea. The enzyme has a pI of 5.93 and is active from pH 4.0 to 10.0, with an optimum at pH 8.0. The purified urease contains nickel and is composed of two protein subunits, with relative molecular masses of 66,000 and 31,000. The subunits were separated and purified and the first 30 N-terminal amino acid residues were determined. A remarkably close relationship was found between both H. pylori urease subunits and jack bean (Canavalia ensiformis) urease, the subunit of which is a single 840 amino acid polypeptide. This subunit is also largely identical to the high molecular mass subunits of the ureases of Klebsiella aerogenes and Proteus mirabilis, evidence that these four ureases are derived from a common ancestral protein.
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PMID:Characterization of the Helicobacter pylori urease and purification of its subunits. 185 97

Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylori was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylori chromosomal DNA which did not lead to urease activity when introduced into E. coli but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.
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PMID:Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. 200 95

Fast protein liquid chromatography and SDS-PAGE have been used to isolate and purify Helicobacter pylori urease. A nickel component of the urease was detected in the purified proteins by atomic absorption spectroscopy. The nickel was present only in the 61 kDa polypeptide and in the ratio of between five and six atoms to one molecule of urease, suggesting a hexameric structure. These results are discussed in relation to other bacterial ureases and urease activity at low pH.
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PMID:The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy. 200 97

The nucleotide sequence of the selA gene from Escherichia coli whose product is involved in the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA) was determined. selA codes for a polypeptide of a calculated Mr of 50,667; a protein of appropriate size was synthesized in vivo in a T7 promoter/polymerase system. An assay for SELA activity was devised which is based on the seryl-tRNA(Sec UCA)-dependent incorporation of [75Se] selenium into acid-insoluble material. It was used to follow SELA purification from cells that overproduced the protein from a phage T7 promoter plasmid. Purified native SELA protein migrates in gel filtration experiments with a native Mr of about 600,000. SELA contains 1 mol of bound pyridoxal 5-phosphate/mol of 50-kDa subunit. Evidence is presented that the overall conversion of seryl-tRNA(Sec UCA) to selenocysteyl-tRNA(Sec UCA) occurs at the SELA protein. SELA, therefore, has the function of a selenocysteine synthase.
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PMID:Selenocysteine synthase from Escherichia coli. Nucleotide sequence of the gene (selA) and purification of the protein. 200 84

A fragment of chromosomal DNA from proteus vulgaris encoding urease was cloned and expressed in Escherichia coli. A 3 kbp region was sequenced and revealed three open reading frames with homology to jack bean (Canavalia ensiformis) urease. The smallest protein (11 kDa) was homologous to the N-terminus of the plant enzyme and the largest polypeptide (61 kDa) corresponded to the C-terminus. The large protein contained conserved regions and a cysteine residue which is known to be catalytically important in the plant enzyme. A protein of 12 kDa showed homology to residues 132 to 237 of jack bean urease.
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PMID:Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes. 218 82

Nucleotide sequence analysis of a Ureaplasma urealyticum DNA fragment, homologous to cloned urease genes of other prokaryotes, revealed three consecutive open reading frames. The molecular weights of the three deduced polypeptides are 11.2 kD, 13.6 kD and 66.6 kD. These values are consistent with the size of the three subunits previously reported for purified native urease. A significant sequence homology was found between the three polypeptides of the ureaplasmal urease and the single polypeptide of jack bean (Canavalia ensiformis) urease. Codon usage indicates that UGA is a tryptophan codon in this mollicute. Use of polymerase chain reactions has disclosed the existence of genetic polymorphism among the urease genes of different serotypes of U. urealyticum.
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PMID:Ureaplasma urealyticum urease genes; use of a UGA tryptophan codon. 219 Nov 84


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