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)

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.
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PMID:Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida. 220

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.
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PMID:Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus. 220 95

The availability of the primary amino acid sequences of the E2 of PDC, alpha-KGDC and BCKADC from several prokaryotic and eukaryotic species has allowed us to compare the structural aspects of human PDC-E2 with those of the E2 components from the other complexes. The PDC-E2 components from all the species examined so far contain three structurally identifiable regions: the lipoyl-bearing domain, the E3-binding site, and the catalytic domain. The primary structure of the lipoyl-bearing domain shows considerable variation in its size, ranging from one to three repeating units of approximately 110 amino acids, but essentially preserving its function in the E2 components. In contrast, the sizes of the E3-binding site and the catalytic domain of PDC-E2 from several species are essentially similar and show considerable conservation of specific amino acid residues. Obviously, additional studies are warranted to better understand the structure-function relationships of these domains and the evolutionary conservation of PDC-E2 in different species. Similarly, the availability of the primary amino acid sequences of E3 from several prokaryotes and eukaryotes has also permitted comparison of the structural domains of these proteins with that of the known structure of human GR, a flavoprotein member of the pyridine nucleotide-disulfide oxidoreductase family. Four structural domains (FAD, NAD+, central, and interface domains) have been identified in the E3 components. On the basis of the comparison of the secondary structural elements of GR and E3, the core structure of these two proteins are shown to be similar. It is hoped that further analysis of E3 using site-directed mutagenesis and determination of its crystal structure will provide better insight into its structure-function relationships.
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PMID:Molecular biology of the human pyruvate dehydrogenase complex: structural aspects of the E2 and E3 components. 248 72

1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol.
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PMID:A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath). 249 29

Anaerobic addition of 0.5 eq of NADH/FAD to the streptococcal NADH oxidase produces a redox form spectrally similar to that obtained with 0.5 eq of dithionite/FAD. The second phase of the titration, however, in addition to reducing the flavin with 1 eq of NADH/FAD, leads to the appearance of a long-wavelength absorbance band centered at 725 nm. Reductive titrations of the enzyme with 3-acetylpyridine-adenine dinucleotide, which has a redox potential 72 mV more positive than that of NADH, yield a similar reduced enzyme species. Dithionite reduction of the NADH oxidase followed by titration with NAD+ partially mimics the long-wavelength absorbance of the NADH-reduced enzyme but also leads to the oxidation of 1 FADH2/dimer. NADH is not formed, however, and a similar result is obtained when the dithionite-reduced oxidase is titrated with the nonreducible substrate analog 3-aminopyridine-adenine dinucleotide. These data indicate that the FADH2 oxidation observed is intramolecular and suggest that the active centers of the two apparently identical subunits/dimer are not equivalent. These results also demonstrate that bound pyridine nucleotides can modulate the redox manifold of the NADH oxidase and, when taken together with the effects of these ligands on pre-steady-state behavior, suggest an important regulatory aspect of the catalytic redox function of this unique flavoprotein.
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PMID:The streptococcal flavoprotein NADH oxidase. II. Interactions of pyridine nucleotides with reduced and oxidized enzyme forms. 251 Nov 96

In dialyzed bovine brain cytosol, the enzymatic formation of nitrogen oxides was directly determined. The basal formation of nitrite and nitrate was concentration-dependently enhanced by L-arginine (EC50 about 3.10(-5) M). Both the basal and L-arginine induced formations were inhibited by NG-monomethyl-L-arginine (EC50 about 2.10(-4) M). In the presence of L-arginine, a concomitant formation of citrulline was detected. L-Arginine methyl ester also served as a substrate, but neither D-arginine, D-arginine methyl ester nor N alpha-benzoyl-L-arginine ethyl ester did so. The formation of nitrite and nitrate was time-dependent, increased linearly with the protein concentration of the cytosol and was not observed when the cytosolic proteins were heat-denaturated. Exogenous NADPH (or NADP+) concentration-dependently enhanced the formation of nitrite and nitrate, whereas NADH, NAD+, FAD, Ca2+, Mg2+ and calmodulin were ineffective. These results indicate that bovine brain contains a cytosolic enzyme which uses NADPH or NADP+ as cofactors to form nitrogen oxides from both an endogenous non-dialyzable substrate and from L-arginine.
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PMID:Enzymatic formation of nitrogen oxides from L-arginine in bovine brain cytosol. 259 Feb 27

Effects of methotrexate (MTX) on mitochondrial oxidative metabolism and ion transport were studied. MTX decreases the membrane potential (delta psi) upon energization of the mitochondrial membrane by NAD+-linked substrates and decreases the amplitude and velocity of swelling induced by glutamate and alpha-ketoglutarate. MTX also has an inhibitory effect on the activities of the oxidation enzymes of NAD+-linked substrates without interfering with the oxidation systems of FAD-linked substrates. The effects of MTX could be interpreted as a consequence of a decrease in the ionic conductivity of the mitochondrial inner membrane.
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PMID:Methotrexate: studies on cellular metabolism. II--Effects on mitochondrial oxidative metabolism and ion transport. 276 70

In this study, we have identified a delta 24-unsaturated intermediate involved in the conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid to cholic acid by the peroxisomal fraction of rat liver. An accumulation of this intermediate was observed when NAD+ was omitted from the reaction mixture. The intermediate was isolated by reversed-phase high-pressure liquid chromatography and identified by combined gas-liquid chromatography-mass spectrometry. The peroxisomal fraction was able to catalyze the conversion of the delta 24-unsaturated intermediate to cholic acid in the presence of CoA, ATP, Mg2+ and NAD+. The identification of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid in cholic acid formation supports the proposed reaction mechanism in which the side-chain cleavage of C27-steroids is similar to that of peroxisomal beta-oxidation of fatty acids. This involves an FAD-dependent oxidase acting on 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA.
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PMID:Identification of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-enoic acid as an intermediate in the peroxisomal conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid to cholic acid. 293 Jul 67

An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography procedure. The enzyme is metal-free and has a molecular mass of about 51 000 Da and probably consists of a single polypeptide chain. As shown by fluorimetric titration, the prosthetic group is 1 mol FAD/mol protein. The affinity behaviour of the enzyme gives evidence for the existence of a dinucleotide-binding domain capable of binding NADH or FAD. The enzyme is specific for NADH (Km = 4.1 X 10(-5) M), NADPH is not oxidized. O2 is the preferred electron acceptor, in addition FAD and, very slowly, one-electron acceptors are reduced. It is not clear whether the reduction of FAD proceeds through the dinucleotide-binding site or by exchange of the prosthetic group. The stoichiometry of the reaction with O2 corresponds to the consumption of 2 mol NADH/mol O2, and only H2O is formed (2 NADH + 2 H+ + O2----2 NAD+ + 2 H2O). Neither H2O2 nor O2.- is detected as intermediate and H2O2 cannot replace O2 as an oxidant. The enzyme can, mainly in its reduced state, be inhibited by -SH reagents. Spectral data give no evidence for the existence of radical intermediates during reduction. The enzyme can obviously accept more than two electrons/mol. On the basis of these data two possible reaction mechanisms are discussed. A proposal for the biological purpose of the reaction is made.
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PMID:Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis. 308 30

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.
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PMID:Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis. 309 21


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