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)

NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.
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PMID:Depression by NAD of x-ray-induced repair-type DNA synthesis in toluene-treated Bacillus subtilis. 16 15

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.
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PMID:Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2. 132 82

D-Amino acid oxidase (EC 1.4.3.3) has been purified from the yeast Trigonopsis variabilis by the application of ion-exchange chromatography on DEAE-cellulose, salt precipitation, gel filtration, and hydroxyapatite adsorption. Alternatively the last two steps can be substituted by a single fast protein liquid chromatographic ion-exchange step (Mono Q). The enzyme appeared homogeneous in PAGE, but small amounts of impurities (not exceeding 5% of total protein) appeared in sodium dodecyl sulfate (SDS)-PAGE. Its Mr in SDS-PAGE is 39,000; it exhibits an isoelectric point of 4.8 and contains 7% (w/v) covalently bound carbohydrate. Its absorption spectrum is similar to hog kidney D-amino acid oxidase, indicating the presence of bound FAD, which, however, could not be separated from the enzyme under non-denaturing conditions. The enzyme is inhibited by SH-oxidizing agents, but not by metal-chelate formers and not by benzoate or toluene. It uses O2 exclusively as the only H acceptor. Km and Vmax values were determined for 15 D-amino acids, which, among 23 tested, were substrates of the enzyme. The enzyme has highest affinity for D-phenylalanine and D-leucine, but maximal activity is obtained with D-citrulline and D-isoleucine. The specific activity of the purified preparation is even higher than that of the commercially available hog kidney enzyme (21.7 vs 16 U/mg). The yeast enzyme may be a useful analytical and preparative tool in view of the difference between its substrate specificity and that of the hog enzyme.
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PMID:D-amino acid oxidase from the yeast Trigonopsis variabilis. 286 42

The reduction of nitrogroups of 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds under the influence of Pseudomonas denitrificans and Escherichia coli enzymes was investigated. Quantitative measurements of the immediate stable products of TNT reduction showed that its reducing attack increased with bacterial age. However, preferential reduction of the nitrogroup in position 2 was typical of bacterial cells of any age, being maintained at pH 5.5 to 7.8. The bacterial reduction in vitro was NAD(P)H- (or NAD(P)H-generating systems) dependent. The nitroreduction was stimulated by only FAD and Mn2+ and Mg2+ out of flavins and metal ions tested. The lack of substrate specificity in the nitroreductases under study is indicated by the reducing capacity (in descending order) of nitroderivatives of benzene, toluene, benzoate, and phenol.
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PMID:[Characteristics of nitroreduction as the key stage in the microbial destruction of aromatic nitro compounds]. 635 6

Cells of Pseudomonas putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methyl-cyclohexa-3,5-diene. One of these components has been purified to homogeneity and shown to be a flavoprotein that contains FAD as the only detectable prosthetic group. Fad was removed from the enzyme during purification. However, equilibrium dialysis experiments showed that the enzyme can bind one mol of FAD/mol of enzyme protein. The apparent molecular weight of the enzyme is 46,000, as judged by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. The latter result suggests the presence of a single polypeptide chain. The amino acid composition of the enzyme reveals a relatively high content of the hydrophobic amino acids leucine, isoleucine, and valine and is remarkably similar in composition to the flavoproteins that function in certain monooxygenase enzyme systems. The purified enzyme catalyzes the reduction of dichloroindophenol, nitrobluetetrazolium, ferricyanide, and ferredoxinTOL. Its ability to reduce cytochrome c and to function in the toluene dioxygenase enzyme system is absolutely dependent on the presence of ferredoxinTOL.
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PMID:Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida. 720 73

Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2 component contains two binuclear iron centers. In total, the structural, spectroscopic, and catalytic features of toluene 2-monooxygenase are reminiscent of soluble methane monooxygenase obtained from methanotrophic bacteria. The two enzyme systems also differ in many subtle ways; for example, they oxidize toluene with completely different regiospecificity.
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PMID:Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4. 757 4

Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.
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PMID:Radioenzymatic assay for reductive catalysis of N(5)N(10)-methylenetetrahydrofolate by methylenetetrahydrofolate reductase. 1108 90

The first intermediate of anaerobic toluene catabolism, (R)-benzylsuccinate, is formed by enzymic addition of the methyl group of toluene to a fumarate cosubstrate and is subsequently activated to (R)-2-benzylsuccinyl-CoA. This compound is then oxidised to benzoyl-CoA and succinyl-CoA by a specific beta-oxidation pathway. The enzyme catalysing the first oxidation step of this pathway, (R)-benzylsuccinyl-CoA dehydrogenase, is encoded by the bbsG gene in Thauera aromatica. It was functionally overproduced in Escherichia coli, purified and characterised. The enzyme is a homotetramer with a subunit size of 45 kDa and contains one FAD per subunit. It is highly specific for (R)-benzylsuccinyl-CoA and is inhibited by (S)-benzylsuccinyl-CoA. An apparent K(m) value of 110+/-10 micro M was obtained for (R)-benzylsuccinyl-CoA. The reaction product of (R)-benzylsuccinyl-CoA dehydrogenase was identified as (E)-benzylidene-succinyl-CoA by comparison with the chemically synthesised compound, which was obtained via a new synthetic procedure. (R)-Benzylsuccinyl-CoA dehydrogenase was detected as a specifically substrate-induced protein in toluene- and m-xylene-grown cells of several bacterial species, using enzyme activity and immunological detection.
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PMID:(R)-Benzylsuccinyl-CoA dehydrogenase of Thauera aromatica, an enzyme of the anaerobic toluene catabolic pathway. 1242 Jan 74

This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.
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PMID:Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex. 1242 69

Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.
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PMID:Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase. 1796 5


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