Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Methylene blue interacts with xanthine oxidase at the iron-sulfide site in the electron pathway (Scheme I) that is known to serve as an electron-sink connecting the reductive and oxidative sites in both the oxidase and dehydrogenase forms. Thus, shunting of electrons to methylene blue at this site effectively diverts their flow away from the FAD site where molecular oxygen is converted to superoxide radicals. Since the electron affinity constants of xanthine oxidase for electron acceptors are FAD greater than iron/sulfide greater than molybdenum, methylene blue falls between the FAD and iron-sulfide site. Thus, methylene blue effectively inhibits superoxide and hydroxyl radical production while accelerating the conversion of xanthine to uric acid. As methylene blue is already approved for medicinal use in humans and is relatively nontoxic, the drug may have a role in reducing tissue injury associated with reperfusion. We are currently investigating this possibility in animal models.
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PMID:Potential of methylene blue to block oxygen radical generation in reperfusion injury. 285 11

Recent studies using magnetic circular dichroism at cryogenic temperatures, electron paramagnetic resonance (EPR) and linear electric field effect-EPR (LEFE) of succinate dehydrogenase in membranes and in soluble, homogeneous preparations demonstrated the presence of 3 different Fe-S clusters in the mammalian enzyme, as well as in a similar bacterial enzyme, fumarate reductase from Escherichia coli. There is one each of the 2Fe, 3Fe, and 4Fe clusters. Thus, succinate dehydrogenase is the first enzyme which has been shown to contain all 3 of these Fe-S clusters. The enzyme also contains 1 mol 8 alpha-[N(3)-histidyl]-FAD. It has taken the combined expertise of many laboratories and 15 years of effort to identify the flavin component, and nearly 3 decades to identify the Fe-S clusters. The data from physical methods appear to be internally consistent, in harmony with the results of chemical analysis, and provide a rational explanation for earlier results by the cluster extrusion method. There remains, however, a number of interesting and substantive questions for future investigations. This review traces the tortuous path, the many pitfalls and false leads, which have led us from the discovery of nonheme iron and 'bound' flavin in the enzyme to elucidation of their structures.
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PMID:The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identification. 299 33

Anaerobic cytochrome c552 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a mutant of Escherichia coli K 12 that synthesizes an increased amount of this pigment. Several molecular and enzymatic properties of the cytochrome were investigated. Its relative molecular mass was determined to be 69 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It was found to be an acidic protein that existed in the monomeric form in the native state. From its heme and iron contents, it was concluded to be a hexaheme protein containing six moles of heme c/mole protein. The amino-acid composition and other properties of the purified cytochrome c552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c552 showed nitrite and hydroxylamine reductase activities with benzyl viologen as an artificial electron donor. It catalyzed the reduction of nitrite to ammonia in a six-electron transfer. FMN and FAD also served as electron donors for the nitrite reduction. The apparent Michaelis constants for nitrite and hydroxylamine were 110 microM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c552 was inhibited effectively by cupric ion and cyanide.
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PMID:Purification of a hexaheme cytochrome c552 from Escherichia coli K 12 and its properties as a nitrite reductase. 300 98

Fumarate reductase was isolated and purified 100-fold to homogeneity from Desulfovibrio multispirans, a new species of sulfate-reducing bacteria. The enzyme contained 1 mol of non-covalently bound FAD and four subunits with Mr 45,000, 32,000, 30,000 and 27,000. EPR spectroscopy showed the existence of two iron-sulfur clusters. The absorption spectrum showed a broad region of high absorbance from 450 nm to 300 nm with a protein peak at 278 nm. The ratio of A278:A400 was 2.60. The specific activity was 110 mumoles H2/mg of protein. The Km for fumarate was 2.5 mM. The activation energy was 8.7 kcal/mol. Electron transport from H2 to fumarate in intact cells was inhibited by 2-heptyl-4-hydroxy-quinoline-N-oxide, a quinone inhibitor, indicating the participation of quinone (probably menaquinone) in fumarate reduction.
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PMID:Isolation of fumarate reductase from Desulfovibrio multispirans, a sulfate reducing bacterium. 300 34

Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources.
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PMID:Glutamate synthase from Bacillus subtilis PCI 219. 301 66

Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.
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PMID:Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme. 302 Dec 12

An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant (S20,W) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe2S2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase activity.
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PMID:Methane monooxygenase: purification and properties of flavoprotein component. 302 58

D-alanine dehydrogenase, an inducible, membrane associated enzyme of Pseudomonas aeruginosa was solubilized from envelope preparations by treatment with Triton X-100 and purified 31-fold in the presence of 0.05% Triton X-100 to 60% homogeneity. Gel electrophoresis indicated the presence of a single subunit of approximately 49,000 molecular weight. The enzyme contained FAD, and absorption spectra were typical of an iron-sulfur flavoprotein. Solubilization produced significant changes in some properties of the enzyme: solubilized enzyme showed increased affinity for D-alanine; a broader substrate specificity; and increased temperature sensitivity, compared with the membrane associated form.
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PMID:Solubilization, purification and characterization of D-alanine dehydrogenase from Pseudomonas aeruginosa and effects of solubilization on its properties. 310 51

Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.
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PMID:Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis. 311 Jan 54

A methylviologen and 8-hydroxy-5-deazaflavin(F420)-reducing hydrogenase was purified over 800-fold to near homogeneity from the archaebacterium Methanococcus voltae with 10 U mg-1 F420-reducing activity. It is the only hydrogenase in this organism. The enzyme showed Km values of 16 microM for F420 and 1.2 mM for methylviologen. A turnover number of 1050 min-1 was calculated for the minimal active unit. The protein tends to aggregate. The molecular mass of the minimal active unit is 105 kDa. Larger molecules of 745 kDa were regularly observed. The enzyme was resolved into subunits with molecular masses of 55 kDa, 45 kDa, 37 kDa and 27 kDa by SDS/polyacrylamide gel electrophoresis. Reversible conversion of an anionic into an uncharged form was observed by DEAE-cellulose chromatography with concomitant changes in substrate specificities. The methylviologen-reducing activity was heat-resistant up to 65 degrees C and was not affected by antiserum raised against the native enzyme, while F420 reduction was inactivated by both treatments. Nickel and selenium contents were determined as 0.6-0.7 mol each, FAD content as 1 mol and iron as 4.5 mol/mol protein (105 kDa), respectively. Electron micrographs taken from the purified enzyme show ring-shaped molecules of 18 nm diameter, which represent the high-molecular-mass species of the enzyme.
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PMID:Purification and characterization of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae. 312 17


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