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)

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.
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PMID:31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase. 156 69

The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxygenase from Pseudomonas putida F1 and have been named bphA, bphE, bphF, and bphG. From this comparison, biphenyl dioxygenase was found to be a multicomponent enzyme containing a two-subunit iron-sulfur protein, a ferredoxin, and a reductase. Comparison of the large subunit of the iron-sulfur protein and the ferredoxin with other multicomponent dioxygenases identified amino acid sequences similar to Rieske iron-sulfur proteins for binding a [2Fe-2S] cluster. Sequences have also been identified in the reductase component that match the consensus sequence for FAD or NAD binding. Transcription of the biphenyl dioxygenase region was examined, and three transcription initiation sites were identified. Transcription initiating at the site furthest upstream is greatly increased when the LB400 cells are grown on biphenyl as the sole carbon source.
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PMID:Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. 156 21

Pseudomonas KB 740 degrades 2-aminobenzoate aerobically via a chimeric pathway which combines characteristics of anaerobic and aerobic aromatic metabolism. Atypically, 2-aminobenzoyl-CoA is an intermediate, and the activated aromatic acid is not only hydroxylated but also reduced to an alicyclic compound in a single step. The bacterial strain possesses a small plasmid, pKB 740, which carries all essential information of this new pathway. Its total nucleotide sequence was determined. It consists of 8280 bp and contains the genes for the two initial enzymes of the pathway; 2-aminobenzoate-CoA ligase catalyzes the activation of the aromatic acid, and the flavoenzyme 2-aminobenzoyl-CoA monooxygenase/reductase catalyzes the hydroxylation (monooxygenase activity) and subsequent reduction (reductase activity) of the aromatic ring of 2-aminobenzoyl-CoA. Furthermore, five open reading frames (ORF) possibly coding for polypeptides are on the plasmid. Putative promoter sequences were found for two of the ORF. A nucleotide sequence able to form a possible termination loop was located downstream of the gene for 2-aminobenzoyl-CoA monooxygenase/reductase. This gene consists of 2190 bases. The deduced amino acid sequence of the protein (730 residues; calculated molecular mass of the native 729-residue protein, 83,559 Da) contains a consensus sequence for an FAD-binding site at the N-terminus and a possible NAD(P)H-binding site approximately 150 amino acid residues apart from the N-terminus. The monooxygenase/reductase shows low sequence similarity to the flavoprotein salicylate hydroxylase. Functional and evolutionary aspects of this work are discussed.
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PMID:Novel aerobic 2-aminobenzoate metabolism. Nucleotide sequence of the plasmid carrying the gene for the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase in a denitrifying Pseudomonas sp. 163 22

Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.
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PMID:Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system. 165 5

Methylenetetrahydromethanopterin reductase from metanogenic archaebacteria catalyzes the reversible reduction of N5,N10-methylenetetrahydromethanopterin to N5-methyltetrahydromethanopterin with reduced coenzyme F420 as electron donor. The enzyme is involved in methane formation from CO2 and in methanol disproportionation to CO2 and CH4. We report here that the reductase from Methanobacterium thermoautotrophicum specifically binds to Blue Sepharose CL-6B. Binding was competitive with coenzyme F420 rather than with NAD, NADP, FAD, FMN, AMP, ADP and ATP. The reductase could also be desorbed with salt. Based on this property an affinity chromatographic procedure for the purification of the enzyme was developed.
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PMID:Single step purification of methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum by specific binding to blue sepharose CL-6B. 169 53

1. An apo-NADPH-ferredoxin reductase was prepared from holo-NADPH-ferredoxin reductase (EC 1.18.1.2) from bovine adrenocortical mitochondria. 2. Amino acid residues of the apo-reductase were modified selectively, to identify the FAD-binding site of the reductase, with chemical reagents such as diethylpyrocarbonate, 5,5'-dithiobis(2-nitrobenzoate), tetranitromethane, pyridoxal 5'-phosphate, p-nitrophenylglyoxal, diisopropylfluorophosphate and N-bromosuccinimide. The binding of FAD to the apo-reductase was measured as quenching of the fluorescence of FAD caused by the binding between apo-reductase and FAD. The quenching was blocked when the apo-reductase was modified with diethylpyrocarbonate and restored on the addition of hydroxylamine. 3. The blocking of the quenching occurred in a competitive manner as to FAD in the presence of diethylpyrocarbonate. However, when the apo-reductase was modified with 5,5'-dithiobis(2-nitrobenzoate), the blocking of the quenching occurred in a non-competitive manner. 4. These results suggested that a histidyl residue of the apo-reductase is essential for the binding of FAD to the reductase. This was confirmed by amino acid sequencing of the modified apo-reductase.
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PMID:Selective chemical modification of amino acid residues in the flavin adenine dinucleotide binding site of NADPH-ferredoxin reductase. 173 87

The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major increase in P450 reductase protein or activity was detectable over a 3-day period. Together, these studies establish that thyroid hormone regulates P450 reductase expression by pretranslational mechanisms. They also suggest that other regulatory mechanisms, which may involve changes in P450 reductase protein stability and/or changes in the translational efficiency of its mRNA, are likely to occur.
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PMID:Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms. 173 85

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."
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PMID:The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases. 174 31

1. In order to investigate the effect of aging on the erythrocyte glutathione system, total glutathione (GSH), glutathione reductase (GSH-red) and glutathione peroxidase (GSH-px) levels were measured in erythrocytes from 33 young (mean age = 30.5 +/- 9.7 years) and 28 aged (mean age = 68.9 +/- 11.4 years) healthy individuals. 2. GSH was 3.5 +/- 1.8 microM/g Hb for the young group, a value significantly greater (P less than 0.01) than 2.3 +/- 0.9 microM/g Hb found for the aged group. Similarly, GSH-red activity, 5.5 +/- 1.8 IU/g Hb, was higher (P less than 0.05) for the young group than 3.4 +/- 0.9 IU/g Hb found for the aged group. The GSH-px activity levels for the young group, 21.1 +/- 5.9 IU/g Hb, were significantly greater (P less than 0.01) than 12.0 +/- 3.3 IU/g Hb for the aged group. The lower activity detected in the aged group for all of these parameters of the glutathione redox system was not related to low levels of hematocrit or hemoglobin. 3. There was no statistical difference in the activation coefficient (AC) of reductase (+FAD/-FAD) between groups, which seems to indicate that the lower activity of glutathione reductase observed in the aged group was not due to riboflavin deficiency. 4. Additional information is required to determine the mechanisms controlling the glutathione redox system and its role in the aging process.
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PMID:Age-related changes of glutathione content, glutathione reductase and glutathione peroxidase activity of human erythrocytes. 182 59

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.
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PMID:NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst. 184 86


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