UNIPROT:O95477 - FACTA Search

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Query: UNIPROT:O95477 (membrane-bound)
29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The O-methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, which has been previously postulated to be the final reaction in the biosynthesis of ubiquinone was demonstrated in vitro using cell extracts of Escherichia coli. S-Adenosyl-L-methionine was active as the methyl donor for the reaction. The enzyme concerned, S-adenosyl-L-methionine: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone-O-methyltransferase, was partially purified and shown to have a molecular weight of about 50 000 and to require a divalent metal and dithiothreitol for optimal activity in vitro. The methyltransferase was absent from extracts from ubiG- mutants suggesting that the ubiG gene is the structural gene coding for the methyltransferase. The enzyme, although not firmly membrane-bound, showed some affinity for the cell membrane in broken cell preparations and could utilize the benzoquinone substrate when the latter was free or bound to the cell membrane, with about equal efficiency. It is concluded that in vivo, the methyltransferase reaction probably occurs at the internal surface of the cytoplasmic membrane.
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PMID:Membrane-associated reactions in ubiquinone biosynthesis. 2-Octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase. 76 31

A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lase was shown to require Mn2+, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.
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PMID:Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-Octaprenyl-4-hydroxybenzoate carboxy-lyase. 78 27

The methyltransferase responsible for the conversion of 5-demethylubiquinone-9 to ubiquinone-9 in rat liver mitochondria has been shown to be localized in the inner membrane of rat liver mitochondria. NADH was required to generate the hydroquinone, which was the immediate substrate for methylation. The Km for 5-demethylubiquinone-9 was estimated to be in the range of 60 to 80 nM and the Km for S-adenosylmethionine was found to be 22 micronM. The methyl-transferase was solubilized by Triton X-100, a procedure which inactivated the 5-demethylubiquinone-9 reductase. Dithionite was found to partially substitute for NADH in both membranous and soluble systems. Inhibitors of catechol-O-methyltransferase were not effective inhibitors of 5-demethylubiquinone-9-methyltransferase. In addition, catechol-O-methyltransferase and 5-demethylubiquinone-9-methyltransferase were found to have reciprocal subcellular localizations. It is likely that the hydrophobic side chain of ubiquinone, added to p-hydroxybenzoate in the first biosynthetic step, is required for attachment to the lipid bilayer. This permits subsequent metabolism of the ring system by membrane-bound enzymes, including the final methylation to form ubiquinone-9.
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PMID:5-demethylubiquinone-9-methyltransferase from rat liver mitochondria. Characterization, localization, and solubilization. 86 14

To verify the assumption of a specific and potent drug action on de novo pyrimidine biosynthesis, isolated dihydroorotate dehydrogenase (DHO-DH) (EC 1.3.3.1) was exposed to Brequinar Sodium (6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt, NSC 368 390) (Brequinar). The membrane-bound DHO-DH was purified to apparent homogeneity (25,000-fold) from rat liver mitochondria in six steps via detergent extraction and subsequent chromatography using the dye ligand Matrex Gel Orange A. Using molecular mechanistic studies (MM2) this ligand was found to mimic closely the stereochemical conformation of Brequinar. SDS-PAGE revealed two protein bands for the purified enzyme with apparent molecular masses of 58 (major) and 68 kDa (minor). In vitro, two modes of action of the DHO-DH are possible: (i) acting as a dehydrogenase in the presence of ubiquinone as proximal electron acceptor and (ii) direct reaction with oxygen as oxidase. A novel assay for the measurement of the oxidase activity was adapted using leuco-dichlorofluorescein-diacetate. Inhibition experiments revealed a striking difference in the susceptibility of DHO-dehydrogenase/oxidase to Brequinar: apparent Ki = 6.09 +/- 0.05 (SD) nM (DHO; ubiquinone n = 10), but Ki = 3.10 +/- 0.09 (SD) mM (DHO; O2). Analyses of initial velocity experiments showed non-competitive inhibition of Brequinar with respect to the substrate dihydroorotic acid in both assays (dehydrogenase and oxidase). The inhibitory effect of the latter was compared to that of the competitive inhibitor 5-aza-dihydroorotate (apparent Ki = 15 +/- 0.25 (SD) microM). The present kinetic data on the action of the purified rodent DHO-DH with Brequinar and computer-aided analyses provide a better insight into the drug-enzyme interaction.
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PMID:Differential susceptibility of dihydroorotate dehydrogenase/oxidase to Brequinar Sodium (NSC 368 390) in vitro. 131 36

The localization of ubiquinone has been investigated in phospholipid bilayer vesicles in studies of fluorescence quenching of membrane-bound probes by ubiquinone homologs (Qn, where n is the number of the isoprenoid units of the chain). Fluorescence-quenching data obtained by using a set of anthroylstearate probes, having the fluorophore located at different depths, revealed that ubiquinone-3 is located throughout the whole bilayer thickness. From the bimolecular quenching constants in the membrane, lateral diffusion coefficients in two dimensions were calculated to span values of 10(-7)-10(-6) cm2.s-1. This suggests that ubiquinones laterally diffuse in a very fluid environment. On this basis, it is proposed that their translational diffusion in the bilayer takes place in two dimensions, with the quinone ring oscillating between the two bilayer surfaces within a hydrophobic environment not extending beyond the glycerol region. This model implies that the quinonic head is both settled near the polar surface of the bilayer and buried into the host hydrocarbon interior. This two-site distribution was confirmed for all Qn, except Q0, by their linear dichroism spectra in the bilayers provided by disc-like lyotropic nematic liquid crystals. These spectra also provided detailed information on the preferential orientations of the quinonic head of the different derivatives within the two sites. The mechanism by which the localization and orientation of Qn guest molecules inside the host bilayer is modulated by the isoprenoid chain length is discussed on a thermodynamical basis. Being that Qn is expected to be also widely contained in the highly curved cristae of the mitochondrial inner membrane, by using rod-like lyotropic nematic liquid crystals we searched out effects of the curvature of the host bilayer on those Qn distributions. The linear dichroism measurements reveal that Qn guest molecules are no longer obliged to find a partition between two different types of localizations when the host bilayer is highly curved. In this case all Qn, even the longest Q10, were found to stay parallel to the amphiphilic chains with a single site localization of the head near the polar interface. By the same linear dichroism technique, the local ordering of all Qn derivatives was also evaluated. The order parameters were found to be basically the same for all derivatives. This result is justified on the basis of the relaxation, caused by the surface curvature, of the lateral compression of the host chains.
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PMID:Localization and preferred orientations of ubiquinone homologs in model bilayers. 144 17

Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.
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PMID:cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer. 145 85

Citrate is fermented by Klebsiella pneumoniae to 2 acetate, 0.5 formate and 1.2 CO2. The formation of less than 1 formate and greater than 1 CO2 per citrate can be accounted for by the oxidation of formate to CO2 in order to provide reducing equivalents for the assimilation of citrate into cell carbon. A membrane-bound electron transport chain is apparently involved in NADH synthesis by these cells. The electrons from formate oxidation to CO2 are used to reduce ubiquinone to ubiquinol by membrane-bound formate dehydrogenase and ubiquinol further delivers its electrons to NAD+, if this endergonic reaction is powered by delta mu Na+. The endogenous NADH level of K. pneumoniae cells thus increased in the presence of formate in response to a delta pNa+ greater than -100 mV. NADH formation was completely abolished in the presence of oxygen or after addition of hydroxyquinoline-N-oxide, a specific inhibitor of the Na(+)-translocating NADH:ubiquinone oxidoreductase. The increase of endogenous NADH was dependent on the delta pNa+ applied to the cells. Inverted membrane vesicles of K. pneumoniae catalysed the reduction of NAD+ to NADH with formate as electron donor after application of delta mu Na+ of about 120 mV consisting of delta pNa+ of 60 mV and delta psi of the same magnitude. Neither the delta pNa+ nor the delta psi of this size alone was sufficient to drive the endergonic reaction. Strictly anaerobic conditions were required for NADH formation and hydroxyquinoline-N-oxide completely inactivated the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:NADH formation by Na(+)-coupled reversed electron transfer in Klebsiella pneumoniae. 150 43

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.
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PMID:Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis. 185 94

Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant. The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone. To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes. Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized. The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices. Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions. All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate. Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees. The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.
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PMID:Electron transfer from menaquinol to fumarate. Fumarate reductase anchor polypeptide mutants of Escherichia coli. 224 42

Gluconobacter suboxydans contains membrane-bound D-glucose and alcohol dehydrogenases (GDH and ADH) as the primary dehydrogenases in the respiratory chain. These enzymes are known to be quinoproteins having pyrroloquinoline quinone as the prosthetic group. GDH reduces an artificial electron acceptor, ferricyanide, in the membrane, but not after solubilization with Triton X-100, while ADH can react with the electron acceptor even after solubilization and further purification. In this study, it has been shown that the ferricyanide reductase activity of GDH is restored by adding the supernatant solubilized with Triton X-100 to the residue, and also by incorporation of purified ADH into the membranes of an ADH-deficient strain. G. suboxydans var. alpha. In addition, the ferricyanide reductase activity of GDH was reconstituted in proteoliposomes from GDH, ADH, and ubiquinone-10. Thus, the results indicated that the electron transfer from GDH to ferricyanide was mediated by ubiquinone and ADH. The data also suggest that GDH and ADH transfer electrons mutually via ubiquinone in the respiratory chain.
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PMID:Evidence for electron transfer via ubiquinone between quinoproteins D-glucose dehydrogenase and alcohol dehydrogenase of Gluconobacter suboxydans. 239 47

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