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

---

Query: EC:1.12.7.2 (hydrogenase)
3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The product of the hypB gene, which is required for the maturation of the three [NiFe]hydrogenases of Escherichia coli, is a member of the GTPase family and exhibits a low intrinsic GTPase activity. It was studied whether or not GTP hydrolysis by HypB is coupled to nickel insertion into hydrogenases and to maturation of hydrogenases. Mutations were introduced into the hypB gene at sites expected to code for amino acids involved in guanine-nucleotide binding. Lys117 of G-motif 1, as well as Asp241 of G-motif 4 were substituted by asparagine residues. The purified mutant HypB proteins showed strongly reduced, but still significant, GTPase activity. In the case of [D241N]HypB, the kcat/Km value was lowered by a factor of 85 and the specificity of the enzyme for GTP was apparently lost, with other nucleoside triphosphates including XTP becoming compatible substrates. The decrease in GTPase activity was even more pronounced for [K117N]HypB. To assess the functionality of these HypB proteins in vivo, the wild-type hypB gene in the chromosome of E. coli was replaced by the mutant alleles. The resulting mutant strains BKN117 and BDN241 were affected in hydrogen metabolism under fermentative conditions. BKN117 did not display hydrogenase activity due to a loss of nickel incorporation into the large subunit. BDN241 exhibited a reduction of hydrogenase activity by 44% and only a portion of the hydrogenase 3 large subunit was in the mature nickel-containing form. From these results, it is concluded that GTP hydrolysis catalysed by HypB is an integral process in nickel incorporation into hydrogenases.
...
PMID:GTP hydrolysis by HypB is essential for nickel insertion into hydrogenases of Escherichia coli. 760 Oct 92

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.
...
PMID:The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein. 842 37

The HypB protein from Bradyrhizobium japonicum is a metal-binding GTPase required for hydrogenase expression. In-frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JH delta Eg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JH delta 23H lacking only the N-terminal histidine-rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JH delta 23H was low in comparison to the wild type in 10-50 nM nickel levels, but could be cured to nearly wild-type levels by including 50 microM nickel during the derepression incubation. Studies on strains harbouring the hup promoter-lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild-type promoter activity in 50 microM NiCl2. Further evidence that HypB is required for hup promoter-binding activity was obtained from gel-shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O2, 10% partial pressure H2) HypB was detected, and its expression preceded hydrogenase synthesis by 3-6 h. 63Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild-type strain at all time points tested during the derepression incubation. Wild-type cultures that received nickel during the HypB expression-specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JH delta 23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal-binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps.
...
PMID:The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase. 914 Sep 70

The hydrogenase accessory protein HypB, or nickelin, has two functions in the N(2)-fixing, H(2)-oxidizing bacterium Bradyrhizobium japonicum. One function of HypB involves the mobilization of nickel into hydrogenase. HypB also carries out a nickel storage/sequestering function in B. japonicum, binding nine nickel ions per monomer. Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches. The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation. A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity.
...
PMID:Dual roles of Bradyrhizobium japonicum nickelin protein in nickel storage and GTP-dependent Ni mobilization. 1069 76

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.
...
PMID:Characterization of Helicobacter pylori nickel metabolism accessory proteins needed for maturation of both urease and hydrogenase. 1253 48

The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.
...
PMID:Metal binding activity of the Escherichia coli hydrogenase maturation factor HypB. 1614 21

Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.
...
PMID:Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system. 1651 46

Assembly of active Fe-hydrogenase in the chloroplasts of the green alga Chlamydomonas reinhardtii requires auxiliary maturases, the S-adenosylmethionine-dependent enzymes HydG and HydE and the GTPase HydF. Genes encoding homologous maturases had been found in the genomes of all eubacteria that contain Fe-hydrogenase genes but not yet in any other eukaryote. By means of proteomic analysis, we identified a homologue of HydG in the hydrogenosomes, mitochondrion-related organelles that produce hydrogen under anaerobiosis by the activity of Fe-hydrogenase, in the pathogenic protist Trichomonas vaginalis. Genes encoding two other components of the Hyd system, HydE and HydF, were found in the T. vaginalis genome database. Overexpression of HydG, HydE, and HydF in trichomonads showed that all three proteins are specifically targeted to the hydrogenosomes, the site of Fe-hydrogenase maturation. The results of Neighbor-Net analyses of sequence similarities are consistent with a common eubacterial ancestor of HydG, HydE, and HydF in T. vaginalis and C. reinhardtii, supporting a monophyletic origin of Fe-hydrogenase maturases in the two eukaryotes. Although Fe-hydrogenases exist in only a few eukaryotes, related Narf proteins with different cellular functions are widely distributed. Thus, we propose that the acquisition of Fe-hydrogenases, together with Hyd maturases, occurred once in eukaryotic evolution, followed by the appearance of Narf through gene duplication of the Fe-hydrogenase gene and subsequent loss of the Hyd proteins in eukaryotes in which Fe-hydrogenase function was lost.
...
PMID:Fe-hydrogenase maturases in the hydrogenosomes of Trichomonas vaginalis. 1652 12

The Escherichia coli protein SlyD is a member of the FK-506-binding protein family of peptidylprolyl isomerases. In addition to its peptidylprolyl isomerase domain, SlyD is composed of a molecular chaperone domain and a C-terminal tail rich in potential metal-binding residues. SlyD interacts with the [NiFe]-hydrogenase accessory protein HypB and contributes to nickel insertion during biosynthesis of the hydrogenase metallocenter. This study examines the HypB-SlyD complex and its significance in hydrogenase activation. Protein variants were prepared to delineate the interface between HypB and SlyD. Complex formation requires the HypB linker region located between the high affinity N-terminal Ni(II) site and the GTPase domain of the protein. In the case of SlyD, the deletion of a short loop in the chaperone domain abrogates the interaction with HypB. Mutations in either protein that disrupt complex formation in vitro also result in deficient hydrogenase production in vivo, indicating that the contact between HypB and SlyD is important for hydrogenase maturation. Surprisingly, SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation. Furthermore, a SlyD truncation lacking the C-terminal metal-binding tail still interacts with HypB but is deficient in stimulating metal release and is not functional in vivo. These results suggest that SlyD could activate metal release from HypB during metallation of the [NiFe] hydrogenase.
...
PMID:The role of complex formation between the Escherichia coli hydrogenase accessory factors HypB and SlyD. 1742 34

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.
...
PMID:Zn2+-linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation. 1876 50


1 2 3 4 Next >>

---