Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.12.7.2 (hydrogenase)
 3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl(2) excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.
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PMID:Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity. 1113 34

Nickel enzymes are a relatively new class of metalloenzymes. The seven known nickel enzymes are urease, hydrogenase, CO-dehydrogenase, methyl-coenzyme M reductase, Ni-superoxide dismutase, glyoxalase I and cis-trans isomerase. The requirement for nickel implies the presence of a nickel-processing system, since free transition metals are harmful to the cell. A nickel-processing system involves the recognition and transport of nickel into the cell and the handling of the nickel once it enters the cell until it is inserted into the nickel enzyme. Several mechanisms for nickel transport have been identified and will be reviewed here. Accessory proteins required for the biosynthesis of the nickel active site have been identified. Accessory proteins bind the nickel when it enters the cell and are proposed to assist with the insertion of nickel into the enzyme. The function of the characterized nickel-processing proteins is described, and models for nickel insertion into the nickel enzymes are presented.
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PMID:Nickel-binding proteins. 1121 9

Moderate levels of urease activity (ca. 300 mU mg(-1)) were detected in Rhizobium leguminosarum bv. viciae UPM791 vegetative cells. This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium. Lower levels of urease activity (ca. 100 mU mg(-1)) were detected also in pea bacteroids. A DNA region of ca. 9 kb containing the urease structural genes ( ureA, ureB and ureC), accessory genes ( ureD, ureE, ureF, and ureG), and five additional ORFs ( orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced. Three of these ORFs ( orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities. R. leguminosarum mutant strains carrying Tn 5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids. orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain. A mutant affected in orf287 exhibited normal levels of urease activity in culture cells. Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R. leguminosarum.
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PMID:Characterization of the urease gene cluster from Rhizobium leguminosarum bv. viciae. 1188 82

Nickel has been shown to be an essential trace element involved in the metabolism of several species of bacteria, archea, and plants. In these organisms, nickel is involved in enzymes that catalyze both non-redox (e.g., urease, glyoxalase I) and redox (e.g., hydrogenase, carbon monoxide dehydrogenase, superoxide dismutase) reactions, and proteins involved in the transport, storage, metallocenter assembly, and regulation of nickel concentration have evolved. Studies of structure/function relationships in nickel biochemistry reveal that cysteine ligands are used to stabilize the Ni(III/II) redox couple. Certain nickel compounds have also been shown to be potent human carcinogens. A likely target for carcinogenic nickel is nuclear histone proteins. Here we present X-ray absorption spectroscopic studies of a model Ni peptide designed to help characterize the structure of the nickel complexes formed with histones and place them in the context of nickel structure/function relationships, to gain insights into the molecular mechanism of nickel carcinogenesis.
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PMID:Use of XAS for the elucidation of metal structure and function: applications to nickel biochemistry, molecular toxicology, and carcinogenesis. 1242 16

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

Nickel is an essential nutrient for selected microorganisms where it participates in a variety of cellular processes. Many microbes are capable of sensing cellular nickel ion concentrations and taking up this nutrient via nickel-specific permeases or ATP-binding cassette-type transport systems. The metal ion is specifically incorporated into nickel-dependent enzymes, often via complex assembly processes requiring accessory proteins and additional non-protein components, in some cases accompanied by nucleotide triphosphate hydrolysis. To date, nine nickel-containing enzymes are known: urease, NiFe-hydrogenase, carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase, methyl coenzyme M reductase, certain superoxide dismutases, some glyoxylases, aci-reductone dioxygenase, and methylenediurease. Seven of these enzymes have been structurally characterized, revealing distinct metallocenter environments in each case.
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PMID:Nickel uptake and utilization by microorganisms. 1282 70

Helicobacter pylori synthesizes two nickel-containing enzymes (urease and hydrogenase), both of which are important pathogenesis factors. Among the many accessory proteins needed for maturation of these Ni-enzymes, are two proteins, HypB and UreG, each of which contain a conserved nucleotide-binding domain (GSGKT). To address the role of this domain in the maturation process, site-directed mutations were introduced in both hypB and ureG. The hypB site-directed mutant strain (Lys59 to Ala59) lacked hydrogenase activity and had less than 1% of the parental urease activity. Hydrogenase activity was partially, and urease activity was fully restored in the hypB mutant strain when grown on nickel supplemented media. The hydrogenase activity of the ureG site-directed mutant strain (Lys14 to Ala14) was comparable to that of the parental strain. However, the ureG mutant strain lacked urease activity, and this deficiency could not be suppressed even when the strain was grown on nickel supplemented media. The expression of immunologically detectable HypB and UreG in the mutants was similar to the parental strain. Expression of the UreA and UreB subunits of urease in both the mutants was also normal. Purified UreG parental and mutant (Lys14 to Ala14) proteins had molecular masses of 27 kDa, but possessed negligible GTP hydrolyzing activity.
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PMID:Roles of conserved nucleotide-binding domains in accessory proteins, HypB and UreG, in the maturation of nickel-enzymes required for efficient Helicobacter pylori colonization. 1452 81

Helicobacter pylori possesses a membrane-bound, nickel containing, hydrogen uptake hydrogenase enzyme; its synthesis requires structural as well as accessory proteins, the latter needed for the complete maturation of the enzyme. Our lab previously characterized mutants in the accessory hyp genes, hypA, hypB, hypD and hypF that were all severely affected for hydrogenase activity, and in some cases (hypA and hypB mutants) also affected for urease activity. This finding prompted us to disrupt the two remaining unstudied hyp genes of H. pylori, hypC and hypE, in order to see if the same pleiotropic effect would be observed. In both mutants hydrogenase activity was abolished but urease activity remained unaffected. Addition of 5 microM nickel into the growth medium partially restored the hydrogenase activity in the hypE mutant and to a lesser extent in the hypC mutant. In addition, we also disrupted the genes HP0634 (referred as hydD in the H. pylori 26695 genome database) and HP0635 (whose function was unknown, referred to here as hydE) to address their possible roles in the hydrogenase synthesis/maturation process. In both cases, hydrogenase activities were abolished and addition of nickel could not restore the activity, suggesting that these proteins are involved in the hydrogenase synthesis process rather than in nickel mobilization/insertion steps.
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PMID:Requirement of hydD, hydE, hypC and hypE genes for hydrogenase activity in Helicobacter pylori. 1472 33

The hyp operon encodes accessory proteins that are required for the maturation of the [NiFe] hydrogenase enzymes and, in some organisms, for the production of urease enzymes as well. HypA or a homologous protein is required for nickel insertion into the hydrogenase precursor proteins. In this study, recombinant HypA from Escherichia coli was purified and characterized in vitro. Metal analysis was used to demonstrate that HypA simultaneously binds stoichiometric Zn(2+) and stoichiometric Ni(2+). Competition experiments with a metallochromic indicator reveal that HypA binds zinc with nanomolar affinity. Spectroscopic analysis of cobalt-containing HypA provides evidence for a tetrathiolate coordination sphere, suggesting that the zinc site has a structural role. In addition, HypA can exist as several oligomeric complexes and the zinc content modulates the quaternary structure of the protein. Fluorescence titration experiments demonstrate that HypA binds nickel with micromolar affinity and that the presence of zinc does not dramatically affect the nickel-binding activity. Finally, complex formation between HypA and HypB, another accessory protein required for nickel insertion, was observed. These experiments suggest that HypA is an architectural component of the hydrogenase metallocenter assembly pathway and that it may also have a direct role in the delivery of nickel to the hydrogenase large subunit.
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PMID:Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. 1599 83

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, expresses a nickel-containing hydrogen-oxidizing hydrogenase enzyme. Growth of a hyaB gene-targeted mutant was unaffected by the presence of hydrogen, unlike the wild-type strain, which showed an enhanced growth rate when supplied with H(2). Hydrogenase activities in H. hepaticus were constitutive and not dependent on the inclusion of H(2) during growth. Addition of nickel during growth significantly stimulated both urease (for wild-type and hyaB) and hydrogenase (for wild-type) activities. In a 5-h period, the extent of (14)C-labeled amino acid uptake by the wild type was markedly enhanced in the presence of hydrogen and was >5-fold greater than that of the hyaB mutant strain. In the presence of H(2), the short-term whole-cell amino acid uptake V(max) of the parent strain was about 2.2-fold greater than for the mutant, but the half-saturation affinity for amino acid transport was the same for the parent and mutant strain. The liver- and cecum-colonizing abilities of the strains was estimated by real-time PCR quantitation of the H. hepaticus-specific cytolethal distending toxin gene and showed similar animal colonization for the hyaB mutant and the wild type. However, at 21 weeks postinoculation, the livers from mice inoculated with wild type exhibited moderate lobular lymphoplasmacytic hepatitis with hepatocytic coagulative necrosis, but the hydrogenase mutants exhibited no histological evidence of lobular inflammation or necrosis.
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PMID:Helicobacter hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and in causing liver lesions in A/J mice. 1611 46


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