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)

Cytochrome P450 102 (BM-3) is a catalytically self-sufficient enzyme from Bacillus megaterium that is presently accepted as an important model of the mammalian microsomal P450 monooxygenase system. We have developed a novel affinity approach to purify P450 102 in a single chromatographic step and have studied the spectroscopic, catalytic, nucleotide binding, and crystallization properties of the highly purified enzyme. B. megaterium ATCC 14581 was grown to high cell density, and P450 102 was purified rapidly and in high yield by chromatography on adenosine-2',5'-diphosphate agarose from crude cell-free extract. The cytochrome bound to the column with remarkable avidity, in contrast to the significantly weaker binding observed for NADPH-cytochrome P450 reductase. Chromatographic behavior also showed that the cytochrome bound NADP(+)-type nucleotides more tightly than any other cellular polypeptide. The purified protein was electrophoretically homogeneous and had essentially theoretical contents of FAD, FMN, and heme. Optical spectra showed the expected heme and flavin absorption bands, and three previously undescribed charge-transfer-type absorptions were characterized. Molar extinction coefficients in the oxidized, fully reduced, and ferrous carbonyl states have been determined; notable is the large soret extinction in the ferrous carbonyl state (epsilon 449 nm = 143,500 M-1 cm-1). Final preparations were active in the oxidation of a wide variety of substrates. Of the C14 alkyl compounds studied, tetradecyltrimethylammonium bromide showed the highest substrate-dependent oxidation of NADPH, followed by myristate and myristyl alcohol; however, myristate exhibited the lowest Km value. Activities were tightly coupled to NADPH oxidation (> 97%). Phenobarbital, benzphetamine, cocaine, cyclohexane, methanol, ethanol, retinoic acid, benzoate, heptaflourobutyrate, and 7-ethoxycoumarin were not substrates. NADP+ titrations showed, as expected, that the coenzyme was bound very tightly, with an average Kd of 580 nM. Our preparations of P450 102 are of sufficient purity and stability that crystals of the native holoenzyme have been grown.
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PMID:Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium. 816 Nov 95

Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells. The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C. The apparent Km and Vmax values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s-1, respectively. The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s-1 using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme. The rate constant kon for substrate binding to the fused enzyme was 25 mM-1.s-1, which is not much different from that of nonfused P4501A1. These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product. On the other hand, the apparent Km and Vmax values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s-1, respectively. The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s-1, respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase. 816 54

Mitochondrial NADPH-linked aquacobalamin reductase was purified and characterized to clarify its enzymatic properties. The enzyme was purified about 360-fold over rat liver mitochondrial membranes in a yield of 7.5%. The purified enzyme was homogenous in SDS-PAGE. The molecular mass (M(r)) of the enzyme was calculated to be 65 kDa by SDS-PAGE and by Toyopearl HW55 gel filtration, indicating that the enzyme is a monomeric polypeptide with M(r) of 65 kDa. The enzyme was a flavoprotein containing 1 mol of FAD and FMN per mole of the enzyme. The enzyme was specific for NADPH as electron donor and had the ability to reduce cytochrome c (15.4 mumol.min-1 x mg protein-1), potassium ferricyanide (4.9 mumol.min-1 x mg protein-1) and 2,6-dichlorophenolindophenol (16.8 mumol.min-1.mg protein-1) as well as aquacobalamin (6.4 mumol.min-1 x mg protein-1). Although the enzyme immunoreacted with an antibody against NADPH-cytochrome P-450 reductase, which had the activity of the NADPH-linked aquacobalamin reductase in rat liver microsomes, the mitochondrial enzyme and the microsomal enzyme had different enzymological properties.
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PMID:Mitochondrial NADPH-linked aquacobalamin reductase is distinct from the NADPH-linked enzyme from microsomal membranes in rat liver. 822 2

Squalene epoxidase is a microsomal membrane-associated enzyme that acts as an important regulator in the sterol biosynthetic pathway. In this study, the enzymatic properties of squalene epoxidase from Saccharomyces cerevisiae were examined. Unlike Candida squalene epoxidase, S. cerevisiae squalene epoxidase required NADPH for enzyme reaction. However, S. cerevisiae enzyme reaction did not require FAD or autologous S105 fraction. Unlike rat squalene epoxidase, the activity of S. cerevisiae was reduced by Triton X-100, a nonionic detergent. Terbinafine, an inhibitor of fungal squalene epoxidase, inhibited the enzyme in a non-competitive manner, while NB-598, an inhibitor of mammalian squalene epoxidase, barely inhibited it in a partially non-competitive manner. Thus, the properties of squalene epoxidase from S. cerevisiae were different from those of squalene epoxidase from rats and Candida, which were previously known. We propose that a species difference of squalene epoxidase exists not only between animals and fungi but between Candida and Saccharomyces.
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PMID:Enzymatic properties of squalene epoxidase from Saccharomyces cerevisiae. 835 82

Molecular properties of superoxide (O2-)-producing cytochrome b558 purified from neutrophils were investigated focusing on the mechanism of the catalytic reaction. The purified cytochrome, which was depleted of FAD, exhibited high O2(-)-generating activity with consumption of NADPH in the presence of microsomal NADPH-cytochrome P-450 reductase. Exogenous additions of CO, CN-, or N3- had no effect on the enzymatic activity. Potentiometric titration of the ferric-ferrous couple of the cytochrome showed that the midpoint reduction potential was -255 mV at pH 7.4. When the reaction of the reduced cytochrome with O2 was analyzed by stopped flow and rapid scanning spectrophotometry, the ferrous form was found to be converted to the ferric form at a rate constant of 9.3 x 10(6) M-1 s-1 at 10 degrees C without showing formation of an oxygenated intermediate. EPR measurement of the ferric cytochrome at 10 K showed that the electronic spin state was in a low spin with g values of 3.2, 2.05, and 1.5. These results suggest that the heme in a six-coordinated low spin state catalyzes one electron reduction of O2 without ligation of O2 to the heme iron during the catalytic cycle.
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PMID:Superoxide-producing cytochrome b. Enzymatic and electron paramagnetic resonance properties of cytochrome b558 purified from neutrophils. 838 86

A microsomal flavin-containing monooxygenase (FMO) was purified 77-fold from macacque liver microsomes on the basis of its methyl p-tolyl sulfoxidase activity. Sequential chromatography on anion- and cation-exchangers, lauryl-Sepharose and 2',5'-ADP-Sepharose provided a purified preparation which exhibited an apparent molecular mass of 59 kDa and a pI of 8.3. N-terminal amino-acid sequencing revealed the characteristic Gly-X-Gly-X-X-Gly consensus sequence for the putative FAD-binding domain of microsomal FMO. In marked contrast to the well-characterized hepatic and pulmonary forms present in experimental animals, the macacque liver enzyme displayed stereoselectivity for sulfoxidation of p-tolyl methyl sulfide on the pro-S rather than the pro-R face of the substrate. Polyclonal antibodies raised against the macacque liver form exhibited little or no cross-reactivity with major purified forms of the enzyme isolated from rabbit liver, guinea-pig liver or rabbit lung. Anti-macacque liver FMO did not cross-react with human fetal liver or adult kidney microsomes, but did recognize a 59 kDa constituent of human adult liver microsomes. The intensity of this immunoreactive 59 kDa band correlated well with human liver microsomal N,N-dimethylaniline N-oxygenase activity. We conclude that human adult liver selectively expresses a microsomal FMO which is functionally and immunochemically distinct from the FMO form(s) present in human fetal liver and adult kidney, and from the major hepatic and pulmonary forms present in common laboratory animals.
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PMID:Purification of macaque liver flavin-containing monooxygenase: a form of the enzyme related immunochemically to an isozyme expressed selectively in adult human liver. 844 76

A cDNA encoding the flavin-containing monooxygenase of rabbit lung (FMO 1B1) was expressed in yeast and Escherichia coli and the recombinant enzymes characterized. A high copy, isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-inducible E. coli expression vector, pKKHC, was used for expression in E. coli strain JM109, and a galactose-inducible vector, YEp53, was used for expression in yeast strain 334. Following transcriptional induction with IPTG or galactose, subcellular fractions were prepared and analyzed immunochemically and catalytically. Antibodies to rabbit FMO 1B1 were used to detect the recombinant proteins in the 100,000 x g pellet prepared from the 10,000 x g supernatant fraction of yeast homogenates and the 2,000 x g supernatant fraction of E. coli homogenates. No FMO 1B1 was detected in cytosol. Mobilities of the recombinant proteins in SDS-polyacrylamide gel electrophoresis appeared identical to that of the native microsomal enzyme. Catalytic similarity to the native FMO 1B1 was demonstrated by the ability of the expressed enzymes to metabolize methimazole, thiourea, dimethylaniline, and cysteamine, but not chlorpromazine or imipramine. In addition, the recombinant enzymes exhibited a number of the unique physical properties associated with FMO 1B1, including stability to elevated temperature and activation by sodium cholate and magnesium chloride. Based on the specific content of FAD, the level of expression was estimated to be approximately 2% of the total protein in the E. coli 100,000 x g particulate fraction and 1% in the fraction from yeast. To demonstrate the utility of the E. coli expression system for studying structure/function relationships of the flavin-containing monooxygenase, two mutant FMOs were expressed and characterized. One mutant, formed by deletion of a putative membrane-anchoring peptide (the 26 carboxyl-terminal amino acids) was tested for membrane association. No difference in the subcellular distribution was found between the truncated and unmodified proteins, suggesting that the 26-residue COOH-terminal peptide is not important in membrane association. Catalytic analysis of the truncated FMO 1B1 established its functional similarity to the full-length protein, indicating that the COOH terminus does not contribute to any of the unique properties of the lung enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains. 844 36

Cytochrome P450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase that, in structural organization and amino acid sequence, resembles the Class II (microsomal) P450 systems. Its single polypeptide chain contains both a P450 heme domain and an NADPH:P450 reductase domain, each of which bears significant homology with its microsomal counterparts. We report here the critical nature of three amino acids in the reductase domain of this enzyme with respect to FMN binding and catalytic activity. We used site-directed mutagenesis to change glycine 570 to bulkier amino acids; none of these mutant enzymes contained FMN after purification. We also made substitutions for tryptophan 574 and tyrosine 536, which by sequence analogy (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158) were proposed to bind FMN through stacking of the aromatic rings with the isoalloxazine ring of the flavin. Mutants of tryptophan 574 which retained the aromatic side chain contained no less than 0.85 mol of FMN per mol of enzyme, while aspartate and glycine substitutions yielded enzymes which did not incorporate FMN. Substitution of tyrosine 536 with aspartate gave an enzyme which contained 0.44 mol of FMN per mol of enzyme but was inactive as a fatty acid hydroxylase and had only 2% of wild-type cytochrome c reductase activity, while the glycine mutant at this position bound no FMN. Furthermore, although all of the mutant enzymes contained 1 mol of FAD per mol of enzyme, the Y536D mutant and those entirely lacking FMN retained no more than 40% of wild-type ferricyanide reductase activity. By assaying these enzymes in the presence of added FMN, we were able to assess the relative importance of the residues in the wild-type sequence with respect to their contribution to FMN binding. In addition, the aromatic mutants of tryptophan 574, which were nearly as active in cytochrome c reduction as wild-type P450BM-3, were only 20% as active in myristate hydroxylation as the wild-type enzyme, suggesting that this amino acid plays an important role in the flow of electrons between the P450 heme and reductase domains.
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PMID:Critical residues involved in FMN binding and catalytic activity in cytochrome P450BM-3. 846 85

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.
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PMID:Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean. 846 4

1. The N-demethylation of mephentermine (MP), p-hydroxymephentermine (p-hydroxy-MP) and N-hydroxymephentermine (N-hydroxy-MP), to produce phentermine (Ph), p-hydroxyphentermine (p-hydroxy-Ph) and N-hydroxyphentermine (N-hydroxy-Ph), and the p-hydroxylation of MP and Ph, to produce p-hydroxy-MP and p-hydroxy-Ph, were examined using rat liver microsomal preparations containing NADPH. Microsomal reduction of N-hydroxy-Ph to Ph was also examined using various cofactors. In addition, enzymic system for the N-demethylation and p-hydroxylation were examined using various inhibitors. 2. N-Hydroxy-MP demethylation to N-hydroxy-Ph proceeded at a rate almost 10-fold faster than other reactions. MP demethylation to Ph, MP oxidation to P-hydroxy-MP, Ph oxidation to p-hydroxy-Ph proceeded at similar rates, whilst p-hydroxy-MP demethylation to p-hydroxy-Ph was catalysed at the slowest rate. Microsomal reduction of N-hydroxy-Ph to Ph required NADH, and the activity was similar to that of MP oxidation to p-hydroxy-MP. 3. N-Demethylation of MP, p-hydroxy-MP and N-hydroxy-MP were inhibited not only by inhibitors of cytochrome P450, but also by methimazole, an inhibitor of the FAD-monooxygenase system. p-Hydroxylations of MP and Ph were inhibited only by inhibitors of cytochrome P450.
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PMID:Metabolism of mephentermine and its derivatives by the microsomal fraction from male Wistar rat livers. 848 60


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