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)

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.
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PMID:NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida. 191 45

The autofluorescence of isolated rat liver cell plasma membranes was characterized in vitro in relation to the autofluorescence used previously for fluorescence recovery after photobleaching (FRAP) studies. The fluorescence of membrane preparations displayed an emission pattern with a maximum at around 525 nm when excited with a 468 nm blue light. The excitation spectrum monitored at 525 nm closely resembled that of flavin compounds (riboflavin, FAD, FMN). The chloroform extract of the membrane fraction showed practically no fluorescence, whereas, both the water-soluble and water-insoluble protein fractions remaining after chloroform extraction were strongly fluorescent. The fluorescence disappeared almost completely under the effect of sodium hydrosulfite, and recovered after oxidation either by shaking in air or by adding buffered hydrogen peroxide solution. The fluorescence of the acid extract of the plasma membranes photolyzed in an alkaline medium was quite similar to that of lumiflavin obtained from the photolysis of riboflavin in an alkaline medium. The plasma membranes prepared from isolated hepatocytes (which were completely devoid of endothelial cell contamination) exhibited the same autofluorescence in the liver cell plasma membranes. The results suggest that the autofluorescence of the liver cell plasma membranes is most likely of a character similar to that of flavin, bound to hepatocyte plasma membrane proteins. This fluorescence is suitable for measuring the average lateral diffusion constant of proteins by means of FRAP methods.
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PMID:Characterization of the autofluorescence of rat liver plasma membranes. 335 26

The products of oxidation of the alpha-tocopherol model compound, 2,2,5,7,8-pentamethyl-6-chromanol (PH) by t-butyl hydroperoxide in chloroform varied with the amount of water present. In the presence of a trace of water, the main products were the spirodimer (PSD) and spirotrimer (PST). As the content of water increased, the main product became 2-(3-hydroxy-3-methylbutyl)-3,5,6-trimethyl-1,4-benzoquinone (PQ). Oxidation of PH in aqueous liposome suspension also produced PQ as the major product. These results suggested that, in aqueous solutions, the major oxidation product of PH would be PQ and of alpha-tocopherol (TH) would be alpha-tocopheryl quinone (TQ). The ease of reduction of PQ and TQ was studied in chemical and biological systems. PQ, TQ, and ubiquinone-10 (UQ) were rapidly reduced to their respective hydroquinones (PQH2, TQH2, and UQH2) at pH 7.3 by NADH plus FAD. Whole blood reduced PQ rapidly at 37 degrees C to PQH2 but did not reduce TQ to TQH2. Human peripheral blood mononuclear cells took up TQ from a bovine serum albumin complex and reduced it to TQH2. Ingestion of TQ (350 mg) by one of us (PSK) resulted in the formation of TQH2 during a 5 h period. These results demonstrate that several biological systems are able to reduce TQ to TQH2 and that it is a reaction that may occur normally in vivo.
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PMID:Is alpha-tocopherol a reservoir for alpha-tocopheryl hydroquinone? 764 91

Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin-NADP+ reductase and, to a lesser degree, with those of NADPH-cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C'(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C'(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.
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PMID:31P-NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C- and 15N-NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states. 969 10

The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 A, alpha = gamma = 90, beta = 110.5 degrees. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 A. Seleno-L-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 A, alpha = gamma = 90, beta = 110.5 degrees and the diffraction limit is 2.7 A.
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PMID:The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa. 1701 92

D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii H(see symbol)-4 using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30 degrees C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33 degrees C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38 degrees C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30 degrees C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. K(m) and V(max) values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform.
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PMID:A simple purification procedure of D-amino-acid oxidase from Candida guilliermondii H(see symbol)-4. 2315 99