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

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neocarzinostatin, a protein antibiotic with anti-tumor activity was found to place single-strand scissions in DNA in an in vitro reaction. The drug's cutting activity was strongly dependent on the presence of 2-mercaptoethanol or dithiothreitol but some cutting did take place in the absence of reducing agent at very high drug levels and prolonged incubation. The requirement for reducing agents could not be replaced with NAD+, FAD, NADH or H2O2 and the strand-scission reaction was not affected by Mg2+, EDTA or intercalating agents. Similar profiles of heat-inactivation of neocarzinostatin were found whether activity was measured by the scission of DNA strand either in vitro or in HeLa cells treated with the drug. Furthermore, both of these parameters corresponded closely with the ability of the modified drug to inhibit DNA synthesis and growth of HeLa cells. By column isoelectric focusing it was shown that all four activities are associated with the same protein band (pH 3.28). From these data we conclude that the cytotoxic activity of neocarzinostatin and the nicking of DNA strands in vitro appear to reside in the same protein.
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PMID:Single-strand nicking of DNA in vitro by neocarzinostatin and its possible relationship to the mechanism of drug action. 13 67

It has been postulated that the peroxisomal fatty acid-oxidizing system [Lazarow & de Duve (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2043--2046; Lazarow (1978) J. Biol. Chem. 253, 1522--1528] resembles that of mitochondria, except for the first oxidative reaction. In this step, O2 would be directly reduced to H2O2 by an oxidase. Two specific procedures developed to detect the activity of the characteristic enzyme fatty acyl-CoA oxidase are presented, namely polarographic detection of palmitoyl-CoA-dependent cyanide-insensitive O2 consumption and palmitoyl-CoA-dependent H2O2 generation coupled to the peroxidation of methanol in an antimycin A-insensitive reaction. Fatty acyl-CoA oxidase activity is stimulated by FAD, which supports the flavoprotein nature postulated for this enzyme. Its activity increases 7-fold per g wet wt. of liver in rats treated with nafenopin, a hypolipidaemic drug. Subcellular fractionation of livers from normal and nafenopin-treated animals provides evidence for its peroxisomal localization. The stoicheiometry for palmitoyl-CoA-dependent O2 consumption, H2O2 generation and NAD+ reduction is 1 : 1 : 1. This suggests that fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal fatty acid-oxidizing system.
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PMID:Detection of peroxisomal fatty acyl-coenzyme A oxidase activity. 51 63

Para-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is one of a group of flavoproteins which insert molecular oxygen into aromatic rings to form phenols. To determine the mechanism of oxygen insertion by this enzyme, an extensive study was made of the reaction with O2 of reduced enzyme in complex with various aromatic molecules. Reactions were studied by following absorbance changes with time with a stopped-flow spectrophotometer. Analysis of multiphasic reactions led to the detection of a minimum of three transient intermediates with characteristic absorption spectra involved in the process of hydroxylation. The initial interaction of oxygen with the reduced enzyme characteristically produces a derivative of FAD (maximum absorbance 380 to 390 nm) which is probably C(4a) peroxyflavin. Depending on the aromatic compound bound to the enzyme, this intermediate decays either to oxidized, enzyme-bound flavin and H2O2 or transfers an atom of oxygen to the aromatic compound. The process of oxygen transfer forms a derivative of FAD of unknown structure (maximum absorbance 390 to 420 nm), which subsequently decays to the third intermediate observed (maximum absorbance 380 to 385 nm), which is probably C(4a) hydroxyflavin. The decay of this last intermediate results in the formation of oxidized enzyme, and the liberation of hydroxylated product and H2O. In an extension of substrate specificity studies it was found that p-aminobenzoate is a substrate and 5-hydroxypicolinate is an effector for p-hydroxybenzoate hydroxylase. The binding of aromatic compounds to the reduced enzyme was observed by following shifts in the absorption spectrum of enzyme bound FADH2, permitting the determination of dissociation constants and kinetics of binding.
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PMID:Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase. 81 94

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens catalyzes in vivo the hydroxylation of p-hydroxybenzoate by molecular oxygen to form 3,4-dihydroxybenzoate. p-Mercaptobenzoate is also a substrate of the enzyme, but instead of being converted to the expected product, 3-hydroxy-4-mercaptobenzoate, the disulfide, 4,4'-dithiobisbenzoate, is formed. To find what mechanistic information this unusual reaction provided, steady state kinetic analyses, combined with rapid reaction studies of the changes in the enzyme-bound FAD, were carried out with the separate half-reactions involved in catalysis. Most of the kinetic measurements were made with a stopped-flow spectrophotometer designed for working anaerobically and connected on line to a minicomputer. Initial rate studies, upon varying systematically the concentrations of p-mercaptobenzoate, NADPH, and oxygen showed that the enzyme interacted with the substrates in the same manner as it does with p-hydroxybenzoate in place of the mercaptan. That is, a ternary complex is formed between enzyme, mercaptobenzoate, and NDAPH, followed by reaction and release of NADP+. Then a second ternary complex is formed between enzyme, mercaptobenzoate, and oxygen followed by reaction, liberation of product, and return to the resting state of the enzyme. Rapid reaction studies showed that the first half-reaction was analagous to that with the natural substrate. The enzyme-flavin is reduced to the 1,5-dihydroflavin by NADPH, and the rate of reaction is dramatically enhanced in the presence of mercaptobenzoate. The rate enhancement with this enzyme correlates well with the presence of a dianion form of the substrate on the enzyme. Examination of the second half-reaction showed that the reduced flavin on the enzyme formed transient intermediates upon reaction with oxygen, which were analogous to the intermediates in reactions where the enzyme forms an hydroxylated product. The oxidation of p-mercaptobenzoate by H2O2 in free solution resulted in the same disulfide as formed in the enzymatic reaction, only orders of magnitude slower. A sulfenic acid was probably the initial oxidation product from p-mercaptobenzoate, and this reacted very fast, and nonenzymatically, with mercaptobenzoate to form the disulfide and H20. The significance of the enzyme reaction with oxygen when complexed with p-mercaptobenzoate is discussed in relation to the mechanism of hydroxylation.
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PMID:Catalytic mechanism of p-hydroxybenzoate hydroxylase with p-mercaptobenzoate as substrate. 82 28

Orcinol hydroxylase (EC 1.14.13.6), which catalyzes the first reaction of orcinol catabolism in Pseudomonas putida 01, has been purified to homogeneity, and crystallized. Orcinol hydroxylase catalyzes the hydroxylation of orcinol with equimolar consumption of O2 and NADH (or NADPH) to 2, 3, 5-trihydroxytoluene, which is nonenzymically oxidized to a quinone. The visible absorption spectrum of the enzyme shows maxima at 373 and 454 nm and a shoulder at 480 nm. FAD can be dissociated from the protein. Reconstitution of enzymic activity was achieved with FAD, and to a limited extent by FMN. The enzyme has a molecular weight of 63,000 to 68,000 and contains 1 mol of FAD per mol of protein. K-m values for the three substrates orcinol, NADH, and O2 are 0.03, 0.13, and 0.07mM, RESPECTIVELY. The molecular activity of the crystalline enzyme is 1560 min minus 1. In the absence of orcinol, NADH is only slowly oxidized with formation of H2O2. Several analogs of orcinol also serve as substrates for hydroxylation, namely resorcinol, 4-methylresorcinol, and 4-bromoresorcinol. Other analogs, m-cresol, m-ethylphenol, 4-ethylresorcinol, and phloroglucinol, mimic orcinol as effectors, in that they (a) accelerate electron flow from NADH to the flavin and (b) decrease the apparent K-m for NADH but not to the same extent as the substrates that are hydroxylated. The latter compounds are not hydroxylated. Instead H2O2 accumulates as the only product of O2 reduction. The enzyme therefore behaves either as a hydroxylase or an oxidase. The ratio of hydroxylase to oxidase activities of the enzyme is decreased by an increase in the temperature of incubation; at 60 degrees the reaction with orcinol is almost 50% uncoupled from hydroxylation. The apparent K-m values for the effectors are in good agreement with the D-D values obtained for orcinol, resorcinol, and m-cresol. K-D values were obtained by measurement of the effector-induced perturbations of the visible absorption spectrum of the flavoprotein by difference absorption spectroscopy. The circular dichroism spectrum of orcinol hydroxylase is also altered in the presence of orcinol. The participation of the flavin in the over-all reaction is demonstrated by its rapid reduction under anaerobic conditions by NADH in the presence or orcinol, resorcinol, or m-cresol. Subsequent introduction of oxygen restores the oxidized form and yields H2O2 when m-cresol is the effector, but not when orcinol is the effector. Transfer of reducing equivalents from the reduced flavoprotein to free FAD may also occur. Reduction of orcinol hydroxylase by NADH in the absence of an effector is 10-4-fold slower than in the presence of an effector. The minimal structural requirements for effectors appear to be a 1,3-dihydroxy or 1-alkyl-3-hydorxybenzene, but only the former are substrates for hydroxylation.
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PMID:Metabolism of resorcinylic compounds by bacteria. Purification and properties of orcinol hydroxylase from Pseudomonas putida 01. 112 36

A 37-yr-old woman with nontoxic goiter is presented. The thyroid 131I uptake at 3 and 24 hr were, respectively, 77.1% and 81.4% dose. Thiocyanate discharged 65.5% of the accumulated 131I in 30 min. In vitro organification of iodine in the thyroid homogenate from the patient was impaired and it was restored to normal by the addition of H2O2, glucose, and glucose oxidase system, FAD, or reduced cytochrome b5. Riboflavin, FMN, oxidized cytochrome b5, oxidized or reduced cytochrome c, NAD(H), and NADP(H) were ineffective in the reaction. The microsomal NADH-cytochrome b5 reductase activity was definitely low in the patient's thyroid. It was augmented to a normal level by incubation of the microsomes with FAD for 30 min or more. The activities of thyroid peroxidase, G6-PD, 6-PGD, catalase, protease, and NADPH-cytochrome c reductase were within normal limits. The major thyroid protein was normal thyroglobulin which could be readily iodinated in the presence of H2O2 and horse radish peroxidase. These findings suggest the correlation of an iodide organification defect with a cytochrome b5 reductase deficiency. Administration of high doses of FAD led to the restoration of thyroidal iodide organification mechanism associated with an increased thyroid hormone production and to a marked decrease of the goiter. Riboflavin was given without effect even at a high dosage level. Consequently, it seems likely that the deficient cytochrome b5 reductase activity in this patient is due to a defect in the biosynthesis of FAD, the coenzyme of the reductase, from riboflavin.
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PMID:Deficient cytochrome b5 reductase activity in nontoxic goiter with iodide organification defect. 116 26

p-Hydroxyphenylacetate-3-hydroxylase, an inducible enzyme isolated from the soil bacterium Pseudomonas putida, catalyzes the conversion of p-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. The enzyme requires two protein components: a flavoprotein and a colorless protein referred to as the coupling protein. The flavoprotein alone in the presence of p-hydroxyphenylacetate and substrate analogs catalyzes the wasteful oxidation of NADH with the stoichiometric generation of H2O2. A 1:1 complex of the flavoprotein and coupling protein is required for stoichiometric product formation. Such complex formation also eliminates the nonproductive NADH oxidase activity of the flavoprotein. A new assay measuring the product formation activity of the enzyme was developed using homoprotocatechuate-2,3-dioxygenase, as monitoring the oxidation of NADH was not sufficient to demonstrate enzyme activity. The coupling protein does not seem to have any redox center in it. Thus, this 2-component flavin hydroxylase resembles the other aromatic hydroxylases in that the only redox chromophore present is FAD.
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PMID:p-Hydroxyphenylacetate-3-hydroxylase. A two-protein component enzyme. 146 99

A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence. It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain. The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H2O2. The apparent Km values for NADH and NADPH are 4.14 microM and 14.0 microM (pH 7.2 at room temperature), respectively, with a sixfold greater kcat/Km values for NADH compared to NADPH. The enzyme uses O2 as an electron acceptor in the presence of either FAD, riboflavin 5'-phosphate or riboflavin as cofactor. In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochrome c, p-nitroblue tetrazolium, 2,6-dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles. No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2- as an intermediate. The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80 degrees C.
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PMID:Purification and characterization of a NADH oxidase from the thermophile Thermus thermophilus HB8. 157 5

A high-abundance NADH-oxidizing enzyme (NADH: acceptor oxidoreductase, EC 1.6.99.3) has been identified and isolated from a range of anaerobic extreme thermophiles, including strains of Clostridium thermohydrosulfuricum and Thermoanaerobium brockii. By use of a pseudo-affinity salt-promoted adsorbent, a nearly pure sample was obtained in one step; remaining impurities were separated by ion-exchange. The fully active purified enzyme contains FAD (two molecules per subunit of 75-78 kDa) and iron-sulphur, and is hexameric in its most active form. The reaction with oxygen is a one- or two-electron transfer to produce superoxide radical and H2O2; other acceptors include tetrazolium salts, dichlorophenol-indophenol, menadione and ferricyanide. The role of the enzyme is not clear; it was found not to be NAD:ferredoxin oxidoreductase, which is a major NADH-utilizing enzyme in these organisms.
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PMID:A thermostable NADH oxidase from anaerobic extreme thermophiles. 159 37

The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.
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PMID:Active-site structural comparison of streptococcal NADH peroxidase and NADH oxidase. Reconstitution with artificial flavins. 174 Apr 31


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