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)

Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis.
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PMID:Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand. 955 61

Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are synthesized starting from the apoprotein via several intermediates by the action of accessory proteins. The isolation and biochemical characterization of such intermediates is hampered by their low abundance and their lability. Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Sepharose matrix. The tag was fused to the large subunit of [NiFe] hydrogenase 3 (HycE) of Escherichia coli. No significant influence of the affinity peptide on maturation or activity of the protein was observed when the modified gene was integrated into the chromosome by homologous recombination. A tagged nickel-free precursor form of HycE bound quantitatively to a recombinant StrepTactin Sepharose column. More than 90% pure subunit could be obtained after elution with desthiobiotin. The procedure was shown to be more efficient than purification by immobilized metal affinity chromatography using a N-terminal His-tag. General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed.
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PMID:Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis. 960 45

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to bind intracellular Ni(II) for transfer to urease apoprotein. While native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni, the Ni-binding sites associated with urease activation are internal to the protein as shown by studies involving truncated H144UreE [Brayman and Hausinger (1996) J. Bacteriol. 178, 5410-5416]. Nine potential Ni-binding residues (five His, two Cys, one Asp, and one Tyr) within H144UreE were independently substituted by mutagenesis to determine their roles in metal binding and urease activation. In vivo effects of these substitutions on urease activity were measured in Escherichia coli strains containing the K. aerogenes urease gene cluster with the mutated ureE genes. Several mutational changes led to reductions in specific activity, with substitution of His96 producing urease activity below the level obtained from a ureE deletion mutant. The metal-binding properties of purified variant UreE proteins were characterized by a combination of equilibrium dialysis and UV/visible, EPR, and hyperfine-shifted 1H NMR spectroscopic methods. Ni binding was unaffected for most H144UreE variants, but mutant proteins substituted at His110 or His112 exhibited greatly reduced affinity for Ni and bound one, rather than two, metal ions per dimer. Cys79 was identified as the Cu ligand responsible for the previously observed charge-transfer transition at 370 nm, and His112 also was shown to be associated with this chromophoric site. NMR spectroscopy provided clear evidence that His96 and His110 serve as ligands to Ni or Co. The results from these and other studies, in combination with prior spectroscopic findings for metal-substituted UreE [Colpas et al. (1998) J. Biol. Inorg. Chem. 3, 150-160], allow us to propose that the homodimeric protein possesses two nonidentical metal-binding sites, each symmetrically located at the dimer interface. The first equivalent of added Ni or Co binds via His96 and His112 residues from each subunit of the dimer, and two other N or O donors. Asp111 either functions as a ligand or may affect this site by secondary interactions. The second equivalent of Ni or Co binds via the symmetric pair of His110 residues as well as four other N or O donors. In contrast, the first equivalent of Cu binds via the His110 pair and two other N/O donors, while the second equivalent of Cu binds via the His112 pair and at least one Cys79 residue. UreE sequence comparisons among urease-containing microorganisms reveal that residues His96 and Asp111, associated with the first site of Ni binding, are highly conserved, while the other targeted residues are missing in many cases. Our data are most compatible with one Ni-binding site per dimer being critical for UreE's function as a metallochaperone.
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PMID:Identification of metal-binding residues in the Klebsiella aerogenes urease nickel metallochaperone, UreE. 1019 22

Syntheses of metal-containing enzymes often require the participation of accessory proteins. The roles played by many of these accessory proteins are poorly characterized. Klebsiella aerogenes urease, a nickel-containing enzyme, provides an ideal system to study metallocenter assembly. Here, we describe a method for isolating a complex containing urease apoprotein and the UreD, UreF, and UreG accessory proteins. We demonstrate that urease apoprotein in this complex is activated to near wild-type enzyme levels when incubated with nickel ions and high (approximately 100 mM) concentrations of bicarbonate. Significantly, we also observed nickel-dependent activation at physiologically relevant (approximately 100 microM) bicarbonate levels, but only in the presence of GTP. Based on studies involving a nonhydrolyzable analog of GTP, we conclude that nucleotide hydrolysis, not just binding, is required for this process. The critical nucleotide-binding site was localized to UreG on the basis of experiments using a variant complex. These studies highlight the relevance of the UreD-UreF-UreG-urease apoprotein complex to nickel metallocenter assembly and explain the previously identified in vivo energy requirement for urease activation.
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PMID:GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins. 1050 Jan 43

Urease possesses a dinuclear Ni active site with the protein providing a bridging carbamylated lysine residue as well as an aspartyl and four histidyl ligands. The apoprotein can be activated in vitro by incubation with bicarbonate/CO2 and Ni(II); however, only approximately 15% forms active enzyme (Ni-CO2-ureaseA), with the remainder forming inactive carbamylated Ni-containing protein (Ni-CO2-ureaseB). In the absence of CO2, apoprotein plus Ni(II) forms a distinct inactive Ni-containing species (Ni-urease). The studies described here were carried out to better define the metal-binding sites for the inactive Ni-urease and Ni-CO2-ureaseB species, and to examine the properties of various forms of Co-, Mn-, and Cu-substituted ureases. Xray absorption spectroscopy (XAS) indicated that the two Ni atoms present in the Ni-urease metallocenter are coordinated by an average of two histidines and 3-4 N/O ligands, consistent with binding to the usual enzyme ligands with the lysine carbamate replaced by solvent. Neither XAS nor electronic spectroscopy provided evidence for thiolate ligation in the inactive Ni-containing species. By contrast, comparative studies of Co-CO2-urease and its C319A variant by electronic spectroscopy were consistent with a portion of the two Co being coordinated by Cys319. Whereas the inactive Co-CO2-urease possesses a single histidyl ligand per metal, the species formed using C319A apoprotein more nearly resembles the native metallocenter and exhibits low levels of activity. Activity is also associated with one of two species of Mn-CO2-urease. A crystal structure of the inactive Mn-CO2-urease species shows a metallocenter very similar in structure to that of native urease, but with a disordering of the Asp360 ligand and movement in the Mn-coordinated solvent molecules. Cu(II) was bound to many sites on the protein in addition to the usual metallocenter, but most of the adventitious metal was removed by treatment with EDTA. Cu-treated urease was irreversibly inactivated, even in the C319A variant, and was not further characterized. Metal speciation between Ni, Co, and Mn most affected the higher of two pKa values for urease activity, consistent with this pKa being associated with the metal-bound hydrolytic water molecule. Our results highlight the importance of precisely positioned protein ligands and solvent structure for urease activity.
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PMID:Characterization of metal-substituted Klebsiella aerogenes urease. 1055 81

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni(2+); however, a truncated form of this protein (H144*UreE) binds only 2 Ni(2+) per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni(2+) to spectinomycin-treated Escherichia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni(2+) per dimer with elevated K(d) values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni(2+) per dimer with unperturbed K(d) values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999) Biochemistry 38, 4078-4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni(2+) with k(1) approximately 18 and k(2) approximately 100 M(-1) s(-1), and with dissociation rates k(-1) approximately 3 x 10(-3) and k(-2) approximately 10(-4) s(-1). Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni(2+) do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation.
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PMID:In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE. 1075 63

The activation of metal-containing enzymes often requires the participation of accessory proteins whose roles are poorly understood. In the case of Klebsiella aerogenes urease, a nickel-containing enzyme, metallocenter assembly requires UreD, UreF, and UreG acting as a protein chaperone complex and UreE serving as a nickel metallochaperone. Urease apoprotein within the UreD-UreF-UreG-urease apoprotein complex is activated to wild-type enzyme activity levels under physiologically relevant conditions (100 microM bicarbonate and 20 microM Ni2+) in a process that requires GTP and UreE. The GTP concentration needed for optimal activation is greatly reduced in the presence of UreE compared to that required in its absence. The amount of UreE provided is critical, with maximal activation observed at a concentration equal to that of Ni2+. On the basis of its ability to facilitate urease activation in the presence of chelators, UreE is proposed to play an active role in transferring Ni2+ to urease apoprotein. Studies involving site-directed variants of UreE provide evidence that His96 has a direct role in metal transfer. The results presented here parallel those obtained from previous in vivo studies, demonstrating the relevance of this in vitro system to the cellular metallocenter assembly process.
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PMID:UreE stimulation of GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein complex. 1101 24

The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated. Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB::TnKm or P1ureA::TnMax5) or producing the inactive apoprotein (N6ureG::TnKm), and with urease-positive clones recovered after complementation of N6ureB::TnKm with ureAB. Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry. With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P<.001). With clone K2(3), producing larger amounts of urease than N6, a significant reduction of bacteria load was observed, in comparison with the wild type (P<.001). N6ureG::TnKm showed adherence characteristics similar to those of N6. The role of urease in internalization was not clear. Thus, urease significantly inhibits H. pylori adherence to Kato III cells by a mechanism largely independent of enzymatic activity.
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PMID:Urease prevents adherence of Helicobacter pylori to Kato III gastric epithelial cells. 1147 Nov 1

UreE is proposed to be a metallochaperone that delivers nickel ions to urease during activation of this bacterial virulence factor. Wild-type Klebsiella aerogenes UreE binds approximately six nickel ions per homodimer, whereas H144*UreE (a functional C-terminal truncated variant) was previously reported to bind two. We determined the structure of H144*UreE by multi-wavelength anomalous diffraction and refined it to 1.5 A resolution. The present structure reveals an Hsp40-like peptide-binding domain, an Atx1-like metal-binding domain, and a flexible C terminus. Three metal-binding sites per dimer, defined by structural analysis of Cu-H144*UreE, are on the opposite face of the Atx1-like domain than observed in the copper metallochaperone. One metal bridges the two subunits via the pair of His-96 residues, whereas the other two sites involve metal coordination by His-110 and His-112 within each subunit. In contrast to the copper metallochaperone mechanism involving thiol ligand exchanges between structurally similar chaperones and target proteins, we propose that the Hsp40-like module interacts with urease apoprotein and/or other urease accessory proteins, while the Atx1-like domain delivers histidyl-bound nickel to the urease active site.
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PMID:Crystal structure of Klebsiella aerogenes UreE, a nickel-binding metallochaperone for urease activation. 1159 23

Synthesis of active Klebsiella aerogenes urease requires four accessory proteins to generate, in a GTP-dependent process, a dinuclear nickel active site with the metal ions bridged by a carbamylated lysine residue. The UreD and UreF accessory proteins form stable complexes with urease apoprotein, comprised of UreA, UreB, and UreC. The sites of protein-protein interactions were explored by using homobifunctional amino group-specific chemical cross-linkers with reactive residues being identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) of tryptic peptides. On the basis of studies of the UreABCD complex, UreD is capable of cross-linking with UreB Lys(9), UreB Lys(76), and UreC Lys(401). Furthermore UreD appears to be positioned over UreC Lys(515) according to decreased reactivity of this residue compared with its reactivity in UreD-free apoprotein. Several UreB-UreC and UreC-UreC cross-links also were observed within this complex; e.g. UreB Lys(76) with the UreC amino terminus, UreB Lys(9) with UreC Lys(20), and UreC Lys(515) with UreC Lys(89). These interactions are consistent with the proximate surface locations of these residues observed in the UreABC crystal structure. MALDI-TOF MS analyses of UreABCDF are consistent with a cross-link between the UreF amino terminus and UreB Lys(76). On the basis of an unexpected cross-link between UreB Lys(76) and UreC Lys(382) (distant from each other in the UreABC structure) along with increased side chain reactivities for UreC Lys(515) and Lys(522), UreF is proposed to induce a conformational change within urease that repositions UreB and potentially could increase the accessibility of nickel ions and CO(2) to residues that form the active site.
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PMID:Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease. 1474 31


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