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)

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

The gene coding for the Candida tropicalis NADPH-cytochrome P-450 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis lambda gt11 expression library and colony hybridization of a C. tropicalis genomic library. The C. tropicalis CPR gene produces a 2.35-kilobase mRNA transcript, levels of which were shown to be increased 16-fold in cells grown on tetradecane relative to cells grown on glucose as the sole carbon source. A 3-kilobase DNA fragment was sequenced, including 554 and 397 base pairs of 5'- and 3'-noncoding sequence, respectively. A single open reading frame of 2040 base pairs was identified and predicts a 76,683-Da polypeptide of 680 amino acid residues. The deduced C. tropicalis CPR amino acid sequence was compared with each of the CPR sequences reported from other organisms and invariant residues were identified. Multiple pairwise alignments of divergent members of protein families, previously recognized for their sequence similarities in their respective binding domains for FMN, FAD, and NADPH, have allowed identification of a subset of these invariant residues. From these analyses we infer the importance of 25 of the 680 amino acid residues.
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PMID:Isolation and characterization of the alkane-inducible NADPH-cytochrome P-450 oxidoreductase gene from Candida tropicalis. Identification of invariant residues within similar amino acid sequences of divergent flavoproteins. 211 6

An enzyme (NADPH-dependent diaphorase) present in rat brain microsomes has been solubilised and shown to utilise both nitrobluetetrazolium and cytochrome c as electron acceptors, when reduced by NADPH. The kinetics of the enzyme have been determined using cytochrome c (Km = 1.3 microM), NADPH (Km = 1.4 microM) and the Vmax (4.7 nmol/min/mg solubilised microsome protein). The subunit Mr is approximately 73,000 D and that of the native enzyme is 170,000-180,000 D, indicating that the enzyme is probably a dimer. Evidence is also provided to show that the enzyme is a flavoprotein, and that it has equimolar amounts of FAD and FMN with respect to the subunit concentration. It seems a possibility that the rat brain diaphorase enzyme may be cytochrome P450 reductase, EC 1.6.2.4.
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PMID:Rat brain NADPH-dependent diaphorase. A possible relationship to cytochrome P450 reductase. 313 10

Two soluble flavoproteins, purified from Escherichia coli cytosol and identified as flavodoxin and NADPH-flavodoxin (ferredoxin) reductase (flavodoxin reductase), have been found in combination to support the 17 alpha-hydroxylase activities of heterologously expressed bovine 17 alpha-hydroxylase cytochrome P450 (P450c17). Physical characteristics of the two flavoproteins including absorbance spectra, molecular weights, and amino-terminal sequences are identical with those reported previously for E. coli flavodoxin and flavodoxin reductase. Flavodoxin reductase, possessing FAD as a cofactor, is able to reconstitute P450c17 activities only in the presence of flavodoxin, an FMN-containing protein, and NAD(P)H. Reducing equivalents are utilized more effectively from NADPH than NADH by flavodoxin reductase. E. coli flavodoxin binds P450c17 directly and with relatively high affinity (apparent Ks approximately 0.2 microM) at low ionic strength, as evidenced by a change in spin state of the P450c17 heme iron upon titration with flavodoxin. This apparent spin shift is attenuated at moderate ionic strengths (100-200 mM KCl). In addition, bovine P450c17 binds reversibly to flavodoxin Sepharose in an ionic strength-dependent manner. These data implicate charge pairing as being important for the interaction between flavodoxin and P450c17. We propose that the amino acid sequence similarity between E. coli flavodoxin-flavodoxin reductase and the putative FMN, FAD, and NAD(P)H binding regions of cytochrome P450 reductase provides the basis for the reconstitution of P450c17 activities by this bacterial system.
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PMID:Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities. 796 51

During the last decade, a multitude of experimental arguments have led to the concept that EDRF is nitric oxide (NO), a messenger not only involved in the control of vasomotor tone but also in vascular homeostasis, neuronal and immunological functions. Regardless of its origin, endogenous NO is produced through the conversion of L-arginine to L-citrulline by NO-synthase (NOS) from which several isoforms have recently been isolated, purified and cloned. NOS-type I (isolated from brain) and type III (isolated from endothelial cells) are termed "constitutive-NOS" and produce picomolar levels of NO from which only a small fraction elicits physiological responses. These isoforms are regulated by Ca(2+)-calmodulin with NADPH, FAD/FMN and tetrahydrobiopterin as co-factors and reveal a high degree of homology with the amino-acid sequence of cytochrome P450 reductase within the C-terminal domain. Functionally, neuronal-NOS type I is important in neurotransmission (modulation of NMDA receptor), the central control of vascular homeostasis and possibly learning and memory. In the peripheral nervous system, NOS appears to be linked to nonadrenergic noncholinergic (NANC) neuronal pathways. Endothelial-NOS type III is essential for the control of vascular tone in response to the release of endogenous mediators, although shear stress is the major trigger of endothelial-NOS activity under physiological conditions. NOS-type III also contributes to the prevention of abnormal platelet aggregation. NOS-types II and IV (isolated from macrophages) are Ca(2+)-calmodulin independent and are termed "inducible-NOS" since their activation is only promoted under pathophysiological situations where macrophages exert cytotoxic effects in response to cytokines. In contrast with NOS-types I and III, activation of NOS-type II in these cells induces the formation of nanomolar levels of NO which act as a defense mechanism of the immune system. Dysfunctions of the L-arginine-NO pathway have been characterized in multiple diseases (atherosclerosis, hypertension, diabetes, sepsis, cerebral ischemia, etc) and the design of more selective activators/inhibitors of NOS isoforms is a new challenge for the understanding of their pathophysiology and treatment.
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PMID:Nitric oxide: an ubiquitous messenger. 829 80

P450BM3 is a bacterial fusion protein between a cytochrome P450 fatty acid hydroxylase (CYP102) and an FAD- and FMN-containing flavoprotein homologous to NADPH: cytochrome P450 reductase. It has been shown that incubation of P450BM3 with NADPH in the absence of a fatty acid substrate results in inhibition of hydroxylase activity [Narhi, L. O., & Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169]. We show that laurate-dependent oxidation of NADPH and oxygen consumption are also inhibited under those conditions. The inhibited enzyme is unable to transfer electrons to the heme iron, but reduces artificial electron acceptors such as cytochrome c, 2,6-dichlorophenolindophenol, or ferricyanide. Incubation with these acceptors rapidly restores hydroxylase activity of P450BM3. The active enzyme is able to catalyze the reduction of cytochrome c and hydroxylation of laurate simultaneously. Cytochrome c has no effect on the K(m) and Vmax of laurate hydroxylation. Laurate and other substrates stimulate cytochrome c reduction by 50-70%. Carbon monoxide inhibits hydroxylase activity, but stimulates cytochrome c reduction 3-4 fold and has no effect on the K(m) for cytochrome c. This stimulation requires binding of a substrate at the heme catalytic site. Laurate binding induces conformational changes in the flavoprotein domain as shown by a 2-fold increase of the flavin fluorescence. Inactivation of P450BM3 by NADPH abolishes the stimulation of cytochrome c reduction by laurate and CO. Complete inhibition of hydroxylase activity correlates with complete lack of stimulation of cytochrome c reduction. The results suggest that a specific conformation of the two domains is maintained in the active P450BM3, ensuring high hydroxylase activity. Cytochrome c reductase and hydroxylase activities of P450BM3 involve different sites of interaction with the flavoprotein domain, different catalytic intermediates, and different rate-limiting steps.
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PMID:Functional interactions in cytochrome P450BM3. Fatty acid substrate binding alters electron-transfer properties of the flavoprotein domain. 894 69

NADPH cytochrome P450 reductase binds two flavin cofactors, FMN and FAD, per molecule of reductase. We have developed an assay to quantitate the reduction activity of FMN-bound flavoprotein. This Trislight assay system takes advantage of the ability of photoactivated flavins to release electrons to acceptors. In turn, electrons derived from Tris buffer restore the flavin to the unexcited, ground state which can again undergo photoactivation to release another electron. FMN-bound reductase, supplied with reducing equivalents from a Tris-light electron generating system, reduces ferricyanide at a rate of 1.8 mumol/min/ nmol reductase. Holoreductase in this system is able to catalyze ferricyanide reduction at a rate of 1.6 mumol/ min/nmol reductase, while FAD-bound reductase has no activity. The 8-NH2-FAD and 8-OH-FAD analog-reconstituted FMN-bound reductase catalyzes the reduction of ferricyanide at rates of 0.43 and 0.28 mumol/min/ nmol reductase, respectively. The riboflavin-reconstituted FMN-bound reductase catalyzes ferricyanide reduction at a rate of 1.1 mumol/min/nmol reductase. FAD or its analogs at the concentrations used to reconstitute enzymatic activity do not support the reduction of ferricyanide in the Tris-light system in the absence of reductase protein. The free flavins, i.e., FMN, 8-OH-FAD, 8-NH2-FAD, and riboflavin, are able to support ferricyanide reduction at a rate of 0.40, 0.52, 0.87, and 0.16 mumol/min/nmol flavin, respectively. This is the first report of an enzymatic assay specific for FMN-bound NADPH cytochrome P450 reductase activity in the absence of its FAD cofactor. Moreover, this report describes the use of an assay procedure based on the provision of reducing equivalents by a Tris-light system which may be useful for other flavin redox enzymes in the absence of reduced pyridine nucleotides or biopterin cofactors.
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PMID:Quantitation of FAD-dependent cytochrome P450 reductase activity by photoreduction. 895 38

The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.
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PMID:Flavin mononucleotide-binding domain of the flavoprotein component of the sulfite reductase from Escherichia coli. 915 34

Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 A resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin-NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 A. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.
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PMID:Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. 923 90

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.
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PMID:Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. 950 Dec 15


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