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:3.5.1.5 (urease)
7,257 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Identification of the environmental triggers involved in the expression of virulence genes is a fundamental objective in studies of bacterial pathogens. For uropathogens, urea, found in the urinary tract at concentrations of up to 500 mm, functions as an environmental signal. Urea freely diffuses into the bacterium Providencia stuartii and activates UreR, a member of the AraC family of transcriptional activators. Active UreR promotes transcription of virulence-associated urease genes and alerts the organisms of its immediate milieu. Thus, the UreR.urea complex has a dual role, acting as both a transcriptional activator as well as an environmental sensor. Here, we describe the molecular events associated with activation of gene expression by urea-bound UreR. The K(d) of the urea.UreR binding reaction was measured as 0.2 mm by fluorescence quenching assays, and the shape of the binding curve indicated a single specific urea-binding site on UreR. Histidine residues are critical for urea binding in urease, and therefore to identify the urea-binding site in UreR, five mutant UreR forms were generated with histidine to alanine substitutions. Two of the mutants (UreR(c)) exhibited a constitutive phenotype by both activating transcription and binding to DNA with an increased affinity in the absence of urea. The UreR(c) bound urea with an affinity similar to that of wild-type UreR. We concluded, therefore, that the mutations resulting in constitutive activity were not involved in the UreR.urea interaction. UreR was activated, then, either by binding urea or by histidine to alanine substitutions at one of two positions. Circular dichroism indicated little change in the structure of UreR when activated, and size-exclusion chromatography demonstrated that both rUreR and rUreR(c) were dimers in both the presence and absence of urea. Thus, the structural changes associated with activation are subtle.
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PMID:Urea-dependent signal transduction by the virulence regulator UreR. 1214 87

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the zinc-assistant hydrolysis of N-acyl-D-amino acids. We report here the cloning, expression, and structural-based mutation of the D-aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue D-aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel alpha/beta-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in D-aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization.
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PMID:Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1. 1238 38

Previous studies demonstrated that two accessory proteins, HypA and HypB, play a role in nickel-dependent maturation of both hydrogenase and urease in Helicobacter pylori. Here, the two proteins were purified and characterized. HypA bound two Ni(2+) ions per dimer with positive cooperativity (Hill coefficient, approximately 2.0). The dissociation constants K(1) and K(2) for Ni(2+) were 58 and 1.3 microM, respectively. Studies on purified site-directed mutant proteins in each of the five histidine residues within HypA, revealed that only one histidine residue (His2) is vital for nickel binding. Nuclear magnetic resonance analysis showed that this purified mutant version (H2A) was similar in structure to that of the wild-type HypA protein. A chromosomal site-directed mutant of hypA (in the codon for His2) lacked hydrogenase activity and possessed only 2% of the wild-type urease activity. Purified HypB had a GTPase activity of 5 nmol of GTP hydrolyzed per nmol of HypB per min. Site-directed mutagenesis within the lysine residue in the conserved GTP-binding motif of HypB (Lys59) nearly abolished the GTPase activity of the mutant protein (K59A). In native solution, both HypA and HypB exist as homodimers with molecular masses of 25.8 and 52.4 kDa, respectively. However, a 1:1 molar mixture of HypA plus HypB gave rise to a 43.6-kDa species composed of both proteins. A 43-kDa heterodimeric HypA-HypB complex was also detected by cross-linking. The cross-linked adduct was still observed in the presence of 0.5 mM GTP or 1 microM nickel or when the mutant version of HypA (altered in His2) and HypB (altered in Lys59) were tested. Individually, HypA and HypB formed homodimeric cross-linked adducts. An interaction between HypA and the Hp0868 protein (encoded by the gene downstream of hypA) could not be detected via cross-linking, although such an interaction was predicted by yeast two-hybrid studies. In addition, the phenotype of an insertional mutation within the Hp0868 gene indicated that its presence is not critical for either the urease or the hydrogenase activity.
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PMID:Characterization of Helicobacter pylori nickel metabolism accessory proteins needed for maturation of both urease and hydrogenase. 1253 48

Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides. The best substrate for the enzyme reported thus far is iso-Asp-Leu. Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively. The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers. Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel. A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel. Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285. The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion. The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group. From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond. This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.
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PMID:High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli. 1271 28

The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.
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PMID:Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein. 1289 98

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism.
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PMID:The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily. 1455 61

Proteus mirabilis, a cause of catheter-associated urinary tract infection, relies on several virulence factors to colonize the urinary tract. Among these, urease contributes to the development of urinary stones resulting from the increase in local pH due to urease-mediated hydrolysis of urea to NH(3) and CO(2). UreR, an AraC-like transcriptional activator, activates transcription of the genes encoding the urease subunits and accessory proteins (ureDABCEFG) in the presence of urea. UreR also initiates transcription of its own gene in a urea-inducible manner by binding to the intergenic region between ureR and ureD. The intergenic region contains poly(A) tracts that appear to be the target of H-NS. It has been shown that Escherichia coli and P. mirabilis H-NS acts to repress transcription of ureR in an E. coli model system. It was hypothesized that H-NS represses urease gene expression in the absence of UreR and urea by binding to the intergenic region. To demonstrate this the P. mirabilis hns gene was cloned and the 15.6 kDa H-NS was overexpressed and purified as a myc-His tail fusion. Using a gel shift assay, purified H-NS-myc-His bound preferentially to a 609 bp DNA fragment containing the entire ureR-ureD intergenic region. H-NS and UreR were able to displace each other from the ureR-ureD intergenic region. Circular permutation analysis revealed that the intergenic region is bent. Moreover, H-NS recognizes this curvature, binds the DNA fragment and induces further bending of the DNA as shown by a circular ligation assay. The effects of H-NS, urea and temperature (25 vs 37 degrees C) on urease expression were shown in E. coli containing an hns knockout and P. mirabilis where expression was increased at 37 degrees C. Increased transcription from p(ureR) was seen in the E. coli hns knockout when temperature was increased from 25 to 37 degrees C. These findings suggest H-NS and UreR differentially regulate urease in a negative and positive manner, respectively.
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PMID:Differential regulation of the Proteus mirabilis urease gene cluster by UreR and H-NS. 1466 72

Urease activation is critical to the virulence of many human and animal pathogens. Urease possesses multiple, nickel-containing active sites, and UreE, the only nickel-binding protein among the urease accessory proteins, activates urease by transporting nickel ions. We performed NMR experiments to investigate the solution structure and the nickel-binding properties of Bacillus pasteurii (Bp) UreE. The secondary structures and global folds of BpUreE were determined for its metal-free and nickel-bound forms. The results indicated that no major structural change of BpUreE arises from the nickel binding. In addition to the previously identified nickel-binding site (Gly(97)-Cys(103)), the C-terminal tail region (Lys(141)-His(147)) was confirmed for the first time to be involved in the nickel binding. The C-terminally conserved sequence ((144)GHQH(147)) was confirmed to have an inherent nickel-binding ability. Nickel addition to 1.6 mm subunit, a concentration where BpUreE predominantly forms a tetramer upon the nickel binding, induced a biphasic spectral change consistent with binding of up to at least three nickel ions per tetrameric unit. In contrast, nickel addition to 0.1 mm subunit, a concentration at which the protein is primarily a dimer, caused a monophasic spectral change consistent with more than 1 equivalent per dimeric unit. Combined with the equilibrium dialysis results, which indicated 2.5 nickel equivalents binding per dimer at a micromolar protein concentration, the nickel-binding stoichiometry of BpUreE at a physiological concentration could be three nickel ions per dimer. Altogether, the present results provide the first detailed structural data concerning the nickel-binding properties of intact, wild-type BpUreE in solution.
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PMID:Structural characterization of the nickel-binding properties of Bacillus pasteurii urease accessory protein (Ure)E in solution. 1476 2

Based on structure of the substrate of urease and for the purpose of designing pharmacophore models for urease inhibitors, which could be effective in physiological and pharmacological studies, a series of twenty-five 1,3,4-diazole-2(3H)-thiones-2(3H)-thiones, 1,3,4-diazoles-2(3H)-thiones, and 1,2,4-tri-3-thiones (OSNs) were designed, synthesized, and evaluated for various kinetic parameters of urease inhibition. OSNs inhibited the activity of urease(s) in a concentration dependent fashion. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of inhibition was of pure competitive type for all the 25 compounds. 5-[4-(hydroxy)phenyl]-1,3,4-thiadiazole-2(3H)-thione was found to be the most active one with a Ki value of 2 microM. The Ki values were increased with an increase in substrate concentrations. Apparently, OSNs employ a homologous mechanism of action by exploiting a common transition catalysis state and acting as ligand chelators to form octahedral complexes with the urease enzymes in an orientation-specific mode. The inhibition was slightly potentiated by lower pH and not abolished in the presence of NH2OH (a scavenger of histidine residue). Because of their safe profile in the genotoxic assay, they may be pursued in the near future for human testing
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PMID:Kinetics of novel competitive inhibitors of urease enzymes by a focused library of oxadiazoles/thiadiazoles and triazoles. 1518 88

Aggregation of jack bean urease (JBU) is associated with alterations of its biological properties, notably the ureolytic and entomotoxic activities. We investigated the influence of metals on protein oligomerization and biological properties. Besides protein aggregation, Cu(2+) induces inhibition of both ureolytic and insecticidal activities of JBU. Chemical modification of histidine residues in JBU with diethylpyrocarbonate (DEPC) decreases its affinity for Cu(2+) and inhibits oligomerization induced by this metal. Furthermore, this modification protects the insecticidal properties of JBU from being inactivated by Cu(2+). Although DEPC-treated JBU displayed lower ureolytic activity, the modified protein is less susceptible to inhibition by Cu(2+) when compared to native enzyme. Our findings show that Cu(2+) promotes JBU aggregation and differently of other heavy metals studied here, it apparently inhibits the ureolytic activity by inducing protein polymerization along with blockage of sulfhydryl groups.
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PMID:Effect of chemical modification of histidines on the copper-induced oligomerization of jack bean urease (EC 3.5.1.5). 1568 Sep 2


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