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)

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.
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PMID:Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase. 1759 15

Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resulting in the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygen atom to water. The two electrons required for the reaction are normally provided by another redox active protein, for example cytochrome P450 reductase (CPR) in mammalian endoplasmic reticulum membranes. P450BM-3 from Bacillus megaterium is a widely studied P450 cytochrome in which the P450 is fused naturally to a diflavin reductase homologous to CPR. From the original characterization of the enzyme by Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzyme concentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence of substrate can diminish this inactivation. We therefore carried out enzyme kinetics, cross-linking experiments, and molecular weight determinations that establish that the enzyme is capable of dimerizing in solution. The dimer is the predominant form at higher concentrations under most conditions and is the only form with significant activity. Further experiments selectively knocking out the activity of individual domains with site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that the electron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during a single turnover, traversing from the FAD domain of one molecule into the FMN domain of the other molecule before passing to the heme domain. Analysis of our results combined with other analyses in the literature suggests that the heme domain of either monomer may accept electrons from the reduced FMN domain.
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PMID:Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3. 1790 5

The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.
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PMID:Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases. 1792 91

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP+ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of equilibria among the redox components in this initial electron-transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation-reduction potential of PdR that had been carefully purified of all residual NAD+, E0' = -369 +/- 10 mV at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). After addition of NAD+, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdRox:NAD+ + 2e- --> PdRrd:NAD+, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E0' = -230 +/- 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH-cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD+ binding to oxidized PdR to establish that the PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases.
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PMID:Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex. 1794 48

The basidiomycete Hypholoma sublateritium produces the triterpenoid antitumor clavaric acid, an inhibitor of the human Ras-farnesyl transferase. The H. sublateritium squalene epoxidase gene (erg1) has been cloned and shown to encode a flavoprotein monooxygenase that requires FAD, NADPH, and P450 cofactors. Silencing of the erg1 gene in H. sublateritium using constructions expressed from the gdh promoter of Agaricus bisporus showed that the squalene epoxidase is involved in clavaric acid formation and in ergosterol biosynthesis; silenced expression of erg1 resulted in an ergosterol-dependent phenotype for full growth. Overexpression of erg1 gene resulted in up to 32% to 97% increment of clavaric acid production confirming its involvement in the biosynthesis of this antitumor product. Oxidosqualene (or dioxidosqualene) appears to be the branching point for primary metabolism (sterols) and secondary metabolites in basidiomycetes.
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PMID:A squalene epoxidase is involved in biosynthesis of both the antitumor compound clavaric acid and sterols in the basidiomycete H. sublateritium. 1809 2

Flavin-containing monooxygenases (FMOs) are, after cytochromes P450, the most important monooxygenase system in humans and are involved in xenobiotics metabolism and variability in drug response. The x-ray structure of a soluble prokaryotic FMO from Methylophaga sp. strain SK1 has been solved at 2.6-A resolution and is now the protein of known structure with the highest sequence similarity to human FMOs. The structure possesses a two-domain architecture, with both FAD and NADP(+) well defined by the electron density maps. Biochemical analysis shows that the prokaryotic enzyme shares many functional properties with mammalian FMOs, including substrate specificity and the ability to stabilize the hydroperoxyflavin intermediate that is crucial in substrate oxygenation. On the basis of their location in the structure, the nicotinamide ring and the adjacent ribose of NADP(+) turn out to be an integral part of the catalytic site being actively engaged in the stabilization of the oxygenating intermediate. This feature suggests that NADP(H) has a moonlighting role, in that it adopts two binding modes that allow it to function in both flavin reduction and oxygen reactivity modulation, respectively. We hypothesize that a relative domain rotation is needed to bring NADP(H) to these distinct positions inside the active site. Localization of mutations in human FMO3 that are known to cause trimethylaminuria (fish-odor syndrome) in the elucidated FMO structure provides a structural explanation for their biological effects.
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PMID:Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase. 1844 1

NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.
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PMID:Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450. 1917 35

NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures.
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PMID:Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase. 1925 66

Bacillus megaterium P450 BM3 (BM3) is an NAD(P)H-binding diflavin reductase exhibiting substantial coenzyme specificity for NADPH over NADH. The side chains of serine 965, arginine 966 and lysine 972 in its FAD-binding domain bind the NADPH 2'-phosphate. Optical, kinetic and thermodynamic properties of S965A, R966A and K972A FAD domains were analyzed singly and combined with the FAD-shielding W1046A mutation. Steady-state and stopped-flow kinetic studies demonstrated substantially decreased NADPH affinity versus wild-type (WT) FAD domain (146-fold for the S965A K(d)). Considerable catalytic efficiency increases (the ratio of specificity constants, k(cat)/K(m), for the coenzymes) with NADH were observed for each point mutant over WT (570-fold in K972A), along with increased rates of NADH-dependent FAD reduction (k(lim) elevated 5.2-fold in R966A). In combination with W1046A, considerable (37 to 56-fold) improvements over WT were seen in the k(lim) parameters with NADH for all double mutants. Each 2'-phosphate binding point mutant produced large increases in FAD potential (111 mV in R966A), despite large distances between these residues and the FAD isoalloxazine ring (18-21 A), suggesting long range conformational influences on FAD environment. The W1046A/K972A mutant abolished NADPH selectivity (8340-fold coenzyme selectivity switch towards NADH), with ramifications for BM3's biotechnological exploitation using the cheaper NADH coenzyme.
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PMID:Probing the molecular determinants of coenzyme selectivity in the P450 BM3 FAD/NADPH domain. 1934 91

Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed-function oxidase system that participates in the metabolism of endo- and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD-to-FMN, but not FMN-to-P450, electron transfer. Here, we report the 2.5 A resolution crystal structure of a functionally competent yeast-human chimeric CPR in an open conformation, compatible with FMN-to-P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 A.
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PMID:Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase. 1948 72


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