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)

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.
...
PMID:Purification and properties of hydrogenase from Megasphaera elsdenii. 52 82

Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.
...
PMID:Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii. 124 77

The gene encoding a protein containing a putative [6Fe-6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60,161 Da). The DNA-derived amino acid sequence was partly confirmed by N-terminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C-terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a Cys-Xaa2-Cys-Xaa7-Cys-Xaa5-Cys motif located towards the N-terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high-spin Fe-S clusters (nitrogenase, hydrogenase), with one significant exception: the stretch containing the first four Cys residues spans two submotifs, Cys-Xaa2-Cys and Lys-Gly-Xaa-Cys-Gly, separated by 11 residues, that are also present in high-spin Fe-S cluster containing CO dehydrogenase. Western-blot analysis demonstrates cross-reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate-reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio.
...
PMID:The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio vulgaris (Hildenborough). 133 51

Sequencing the genes encoding the methyl viologen-reducing hydrogenase, cloned from Methanobacterium thermoautotrophicum strain delta H and Methanothermus fervidus, revealed the presence of tightly linked genes, designated mvhB, which were predicted to encode proteins containing six tandemly arranged bacterial ferrodoxin-like domains. A lacZ-mvhB gene fusion has been constructed and expressed in Escherichia coli. Rabbit antibodies raised against the fusion polypeptide purified from E. coli have been used to identify and isolate the polyferrodoxin from Mb. thermoautotrophicum strain delta H. The polyferredoxin accumulates in cells of the methanogen during exponential growth but decreases rapidly on entry into stationary phase. It is not processed into monoferredoxins and is located primarily in the soluble fraction of cell lysates of Mb. thermoautotrophicum. Metronidazole reduction by crude extracts of Mb. thermoautotrophicum strain delta H cells, dependent on the presence of hydrogen and the heterodisulfide CoM-S-S-HTP [formed from the two coenzymes 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) and N-(7-mercaptoheptanoyl)threonine O3-phosphate (HS-HTP)], was not inhibited by the antibodies raised against the LacZ-MvhB fusion polypeptide.
...
PMID:Identification and isolation of the polyferredoxin from Methanobacterium thermoautotrophicum strain delta H. 149 82

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2 = H4MPT) to methenyltetrahydromethanopterin (CH identical to H4MPT+) and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2 = H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH identical to H4MPT+ with 3H2 generating [methylene-3H]CH2 = H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.
...
PMID:H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea. 152 40

In Azotobacter chroococcum the hydrogenase structural genes (hupSL) cover about 2.8 kb of a 15-kb region associated with hydrogen-uptake (Hup) activity. Two other genes in this region, hupD and hupE, were located 8.9 kb downstream of hupL and were shown to be essential for hydrogenase activity by insertion mutagenesis. A fragment of DNA beginning 3.4 kb downstream of hupL was able to complement the hupE mutant, supporting earlier evidence for a promoter downstream of hupSL. Hybridization experiments showed that hupD and hupE share some similarity with a region of Alcaligenes eutrophus DNA which is apparently involved in the formation of catalytically active hydrogenase. The hupD gene encodes a 379-amino acid, 41.4-kDa polypeptide while hupE codes for a 341-amino acid, 36.1-kDa product. The predicted amino acid sequences of the hupD and hupE genes are homologous to the Escherichia coli hypD and hypE gene products, respectively. A polar mutation in hupD had no effect on beta-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupD and hupE are probably not involved in regulating hydrogenase structural gene expression.
...
PMID:Characterization of two genes (hupD and hupE) required for hydrogenase activity in Azotobacter chroococcum. 152 70

The enzymology of the heterodimeric (NiFe) and (NiFeSe) hydrogenases, the monomeric nickel-containing hydrogenases plus the multimeric F420-(NiFe) and NAD(+)-(NiFe) hydrogenases are summarized and discussed in terms of subunit localization of the redox-active nickel and non-heme iron clusters. It is proposed that nickel is ligated solely by amino acid residues of the large subunit and that the non-heme iron clusters are ligated by other cysteine-rich polypeptides encoded in the hydrogenase operons which are not necessarily homologous in either structure or function. Comparison of the hydrogenase operons or putative operons and their hydrogenase genes indicate that the arrangement, number and types of genes in these operons are not conserved among the various types of hydrogenases except for the gene encoding the large subunit. Thus, the presence of the gene for the large subunit is the sole feature common to all known nickel-containing hydrogenases and unites these hydrogenases into a large but diverse gene family. Although the different genes for the large subunits may possess only nominal general derived amino acid homology, all large subunit genes sequenced to date have the sequence R-X-C-X-X-C fully conserved in the amino terminal region of the polypeptide chain and the sequence of D-P-C-X-X-C fully conserved in the carboxyl terminal region. It is proposed that these conserved motifs of amino acids provide the ligands required for the binding of the redox-active nickel. The existing EXAFS (Extended X-ray Absorption Fine Structure) information is summarized and discussed in terms of the numbers and types of ligands to the nickel and the various redox species of nickel defined by EPR spectroscopy. New information concerning the ligands to nickel is presented based on site-directed mutagenesis of the gene encoding the large subunit of the (NiFe) hydrogenase-1 of Escherichia coli. Based on considerations of the biochemical, molecular and biophysical information, ligand environments of the nickel in different redox states of the (NiFe) hydrogenase are proposed.
...
PMID:Structure-function relationships among the nickel-containing hydrogenases. 155 64

Factor F390 is the 8-OH adenylated form of the deazaflavin coenzyme F420, which is a central electron carrier in methanogenic bacteria. The enzymes catalysing the formation of F390 from ATP and F420 (F390 synthetase) and its hydrolysis into AMP and F420 (F390 hydrolase) were isolated and partially purified from Methanobacterium thermoautotrophicum. Both enzymes were oxygen-stable. The F390 synthetase tended to coelute with coenzyme F420 reducing hydrogenase during all purification steps. The 30-fold purified enzyme was still contaminated with the hydrogenase. The F390 hydrolase was purified 135-fold to a specific activity of 8.6 mumol/min/mg protein. The colourless enzyme consisted of one polypeptide of approximately 27,000 kd.
...
PMID:F390 synthetase and F390 hydrolase from Methanobacterium thermoautotrophicum (strain delta H). 164 79

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.
...
PMID:Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus. 184 42

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. The NADH-binding subunit (Mr = 50,000) of this enzyme complex was identified by direct photoaffinity labeling with [32P]NADH [Yagi, T., & Dinh, T.M. (1990) Biochemistry 29, 5515-5520]. Primers were synthesized on the basis of the N-terminal amino acid sequence of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the alpha subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex I were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.
...
PMID:The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: gene cloning and deduced primary structure. 190 52


1 2 3 4 5 6 7 Next >>

---