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: EC:1.1.1.1 (alcohol dehydrogenase)
9,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The NADPH-cytochrome P450 reductase gene isolated from the yeast Saccharomyces cerevisiae [Yabusaki et al., J. Biochem. 103, 1004-1010 (1988)] was expressed on a multi-copy plasmid in the yeast. The transformed yeast cells with the recombinant plasmid carrying the reductase gene with a length of 3 kb produced the corresponding mRNA read from the original transcription initiation site under the control of its own promoter with a maximum length of 300 bp. The reductase content in the transformed cells was 25 times higher than that of the endogenous reductase. When the coding region for the reductase was placed between the alcohol dehydrogenase I gene promoter and the terminator of the expression vector pAAH5, the expression level was 32 times higher than at the endogenous level. These recombinant yeast strains showed enhanced cytochrome c reductase activity with increased cellular reductase levels. A simultaneous expression of yeast P450 reductase with rat P450c or bovine P450(17 alpha) resulted in 25 times or a 5 times increase in the corresponding P450-dependent monooxygenase activity of the recombinant yeast strains, respectively, as compared with that of the yeast cells expressing the corresponding P450 species. These results suggested that the overproduction of yeast P450 reductase with a simultaneous expression of the mammalian P450 species enhanced the P450c- and P450(17 alpha)-dependent monooxygenase activities in the recombinant yeast strains, probably due to the increased frequency of the interaction between yeast P450 reductase and P450c or P450(17 alpha) in the yeast microsomes.
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PMID:Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevisiae. 212 33

Seven P450/reductase fused enzymes were produced in Saccharomyces cerevisiae by expressing fused cDNAs consisting of bovine cytochrome P450c17 (P450c17) and yeast NADPH-cytochrome P450 reductase (reductase). These fused enzymes differed in the length and amino acid sequence of the hinge region between the P450 and reductase moieties. Expression of the fused constructs under the control of the yeast alcohol dehydrogenase I promoter and terminator of expression vector pAAH5 in S. cerevisiae AH22 cells resulted in the production of about 2-8 X 10(4) molecules per cell of the seven corresponding fused enzymes. Six of the fused enzymes incorporated a protoheme, as confirmed by reduced CO-difference spectra. Recombinant yeast strains producing each of the fused hemoproteins showed P450c17-dependent 17 alpha-hydroxylase activity toward progesterone. The most active fused enzyme, delta N23FE, which lacked the amino-terminal 23 amino acids of the reductase, showed about 10 times higher 17 alpha-hydroxylase activity than bovine P450c17, although the fused enzyme (delta N23FE)' with an amino acid sequence in the hinge region different from delta N23FE was less active than delta N23FE. The fused enzyme delta N0FE, consisting of P450c17 and whole reductase, showed about 1.8 times higher activity than bovine P450c17. No activity was found with delta N84FE lacking the amino-terminal 84 amino acids of the reductase moiety. P450c17-dependent C17,(20)-lyase activity toward 17 alpha-hydroxyprogesterone was detected to lesser extents in the recombinant yeast. Fused bovine P450c17/yeast reductase enzymes show enhanced 17 alpha-hydroxylase activity, and the length and amino acid sequence in the hinge region between the P450c17 and yeast reductase moieties can be important for efficient intramolecular electron transfer in the fused enzymes.
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PMID:Genetically engineered P450 monooxygenases: construction of bovine P450c17/yeast reductase fused enzymes. 218 Apr 29

1. A systematic kinetic investigation of the reduction of aryl-nitroso compounds by pyridine and flavin coenzymes and their analogs, in enzymatic and nonenzymatic systems, has been reported. 2. Two main groups of nitroso compounds have been investigated, representatives nitroso-benzene and 1-nitroso-2-naphthol; in all enzymatic and nonenzymatic systems, the former was always reduced to phenyl-hydroxyl-amine and the latter to 1-amino-2-naphthol. 3. Pyridine compounds included NADH, APAD-4H2 and DBNA-4H2 in nonenzymatic systems, and liver alcohol dehydrogenase. Flavin compounds included 1,5-dihydrolumiflavin and various forms of reduced 5-ethyl-lumiflavin, in nonenzymatic systems, and the flavoenzymes glucose-oxidase and NADPH-cytochrome P450 reductase. 5. Pyridine coenzymes and their analogs reduced nitroso compounds by a direct hydride transfer, with a primary kinetic isotope of 9.5 +/- 2.2. 6. All flavin compounds (glucose-oxidase and its nonenzymatic analog 1,5-dihydrolumiflavin and NADPH-cytochrome P450 reductase and its analog 5-ethyl-1,5-dihydrolumiflavin) reduced aryl-nitroso compounds with high efficiency (k2 greater than 10(5)M(-1) min(-1)). 7. The flavin compounds have been shown to be much more efficient reductans of nitroso compounds, compared to pyridine coenzymes, both in enzymatic and nonenzymatic systems; the only exception to this rule presented the extremely efficient reduction of p-substituted aryl-nitroso compounds by liver alcohol dehydrogenase.
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PMID:Reduction of aryl-nitroso compounds by pyridine and flavin coenzymes. 253 Oct 98

cDNA complementary to mRNA coding for a minor form of cytochrome P-450 from phenobarbital-treated rabbit liver (pHP3) was isolated using cDNA for the major phenobarbital-inducible cytochrome P-450 of rat liver as a probe in the first screening of a cDNA library. The nucleotide sequence of pHP3 was determined and contained a continuous reading frame encoding 490 amino acids. The deduced amino acid sequence of pHP3 protein exhibited about 50% homology with the major cytochrome P-450 from phenobarbital-treated rabbit liver, while the homology was as high as 80% between two minor cytochrome forms, pHP2 and pHP3. Two expression plasmids, pAHF3 and pAH delta N3, were constructed by insertion of pHP3 fragment between yeast alcohol dehydrogenase 1 (ADH1) promoter and terminator regions. pAHF3 contained the entire coding sequence of pHP3, but nucleotide sequences for the N-terminal region of pHP3 protein (from the 2nd to the 3rd amino acid) were deleted in pAH delta N3. On introduction of the constructed plasmids into Saccharomyces cerevisiae AH22 cells, the absorption spectrum of cytochrome P-450 was detected in the microsomal fraction from the transformed cells carrying pAHF3. On the other hand, cytochrome P-450 could not be detected spectrophotometrically in any subcellular fractions from the yeast cells carrying pAH delta N3, although the transcript of pHP3 insert was detected in RNA blot analysis. These results suggest that the N-terminal region of pHP3 protein plays an important role in accumulation of the newly synthesized pHP3 protein in yeast cells. Cytochrome P-450 (pHP3) was solubilized from microsomal membranes of the transformed yeast cells and purified partially on an aminooctyl Sepharose column (specific content, about 6 nmol per mg of protein). In the oxidized state the cytochrome preparation exhibited an absorption spectrum characteristic of a low-spin ferric cytochrome P-450. The reduced CO complex of the cytochrome showed a Soret absorption maximum at 450 nm. The monooxygenase activity of cytochrome P-450 (pHP3) was examined in a reconstituted system containing the cytochrome preparation and NADPH-cytochrome P-450 reductase. Cytochrome P-450 (pHP3) catalyzed N-demethylation of benzphetamine and aminopyrine and denitrification of 1-nitropropane. Addition of cytochrome b5 to the reconstituted system resulted in stimulation of the N-demethylation activities but inhibition of the denitrification activity. Neither 7-ethoxycoumarin O-deethylation activity nor acetanilide p-hydroxylation activity was detected, either in the presence or absence of cytochrome b5.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cytochrome P-450 related to P-4504 from phenobarbital-treated rabbit liver: molecular cloning of cDNA and characterization of cytochrome P-450 obtained by its expression in yeast cells. 282 Sep 51

A full-length cDNA for rat NADPH-cytochrome P-450 reductase was cloned by the procedure of Okayama and Berg (1982) from hepatic poly(A)RNA prepared from phenobarbital-induced rats. Both cDNA and amino acid sequences agreed with the sequences reported by Porter and Kasper (1985) except for four single base differences. Three expression plasmids were constructed by insertion of the reductase cDNA between yeast alcohol dehydrogenase I (ADH) promoter and terminator regions. Plasmids pARF1 and pTRF2 were constructed with slightly different lengths between the ADH promoter and the initiation codon; on introduction into Saccharomyces cerevisiae AH22 cells, they synthesized about 1 X 10(3) and 5 X 10(3) reductase protein molecules per cell, respectively. A third plasmid, pARM1, containing a cytochrome P-450MC cDNA expression unit located between two reductase cDNA expression units synthesized 4 X 10(5) cytochrome P-450MC hemoprotein and 1 X 10(4) reductase protein molecules per cell. The cellular extracts of the AH22/pARM1 strain, which synthesized both rat enzymes, showed higher cytochrome c reductase and cytochrome P-450MC-dependent 7-ethoxycoumarin O-deethylation activities as compared to extracts of the AH22/pAMC1 strain, which synthesized only rat cytochrome P-450MC. 7-Ethoxycoumarin O-deethylation activity in the cellular extract of AH22/pARM1 strain was partly inhibited by the addition of anti-rat reductase IgG. In addition, whole AH22/pARM1 cells exhibited higher monooxygenase activity toward acetanilide and 7-ethoxycoumarin than control AH22/pAMC1 cells. These results indicated that a functional electron-transport chain consisting of rat NADPH-cytochrome P-450 reductase and rat cytochrome P-450MC was constructed in S. cerevisiae cells.
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PMID:Expression of rat NADPH-cytochrome P-450 reductase cDNA in Saccharomyces cerevisiae. 308 10

A hybrid cDNA encoding a fused enzyme consisting of rat cytochrome P450c and rat NADPH-cytochrome P450 reductase was constructed by combining the cytochrome P450c cDNA with the cDNA fragment encoding the protease-solubilized moiety of the NADPH-cytochrome P450 reductase. The hybrid cDNA was inserted between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5 to yield expression plasmid pAMP19. Saccharomyces cerevisiae AH22 cells transformed with the expression plasmid pAMP19 produced a 130-kD protein reactive with both anti-cytochrome P450c Ig and antireductase Ig. The yeast cells containing the fused enzyme exhibited about four times higher monooxygenase activity toward 7-ethoxycoumarin than those containing rat cytochrome P450c alone. The fused enzyme was purified from the yeast microsomal fraction by sequential chromatography with DEAE-cellulose and 2',5'-ADP Sepharose 4B columns. The preparation had an apparent molecular weight of 130 kD and the same sequence of the 10 amino-terminal amino acids as that of rat cytochrome P450c. Spectral properties of the fused enzyme indicated the presence of a protoheme, flavin adenine dinucleotide, and flavin mononucleotide in the molecule. The reaction mechanism of the fused enzyme followed first-order kinetics. These results clearly indicate that the fused enzyme is a new self-catalytic P450 monooxygenase. Trypsin treatment of yeast microsomes containing the fused enzyme suggested that the P450 moiety is embedded in the microsomal membrane with the reductase moiety lying on the cytoplasmic side.
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PMID:A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase. 310 64

Modified constructions of a microsomal cytochrome P450, of NADPH-cytochrome P450 reductase, and of a P450/reductase fused enzyme were prepared to analyze the function of the amino-terminal hydrophobic regions of these enzymes and the hinge region of the fused enzyme. Expression plasmids for delta P450c, delta reductase, and the delta P450/reductase fused enzyme, all of which lacked their amino-terminal hydrophobic regions, were constructed by inserting each of the corresponding cDNAs between the yeast alcohol dehydrogenase I promoter and the terminator of the expression vector pAAH5. Yeast transformed with plasmids encoding delta P450 and the delta P450/reductase fused enzyme produced smaller amounts of the respective enzymes and showed lower monooxygenase activity toward 7-ethoxycoumarin than did yeast transformed with plasmids encoding the complete enzymes. Both delta P450 and delta P450/reductase were found in the microsomal fraction of the yeast cells. Yeast transformed with the expression plasmid for delta reductase produced 20 times more enzyme than did yeast transformed with the plasmid for the complete enzyme. delta Reductase was present in the soluble fraction and was 33 times more active in reducing cytochrome c than was the complete enzyme. The results suggest that the amino-terminal hydrophobic regions of P450c and the P450/reductase fused enzyme play an important role in their stability and function in the yeast microsomes. By contrast, the amino-terminal-containing P450 reductase appears to be unstable in yeast cells. Altering the size of the hinge regions does not affect the activity of the P450/reductase fused enzyme significantly, but some amino acid changes in this region increase the stability of the fused enzyme slightly.
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PMID:Genetically engineered modification of P450 monooxygenases: functional analysis of the amino-terminal hydrophobic region and hinge region of the P450/reductase fused enzyme. 314 46

Microsomes isolated from rats treated with either pyrazole or 4-methylpyrazole, potent inhibitors of alcohol dehydrogenase, catalyzed the oxidation of ethanol and 2-butanol at rates 2-3-fold higher than saline controls. Time course experiments and dose-response experiments indicated that an increase in the microsomal oxidation of alcohols could be observed 24 hr after a single treatment with 200 mg/kg body weight of either pyrazole or 4-methylpyrazole, and after 2 or 3 days of treatment with 50 mg/kg of either of these compounds. The pyrazole treatment did not change the activity of NADPH-cytochrome P-450 reductase, the content of cytochrome P-450, or the oxidation of aminopyrine. Hence, microsomal oxidation of alcohols was increased by the pyrazole treatment whether results were expressed "per mg of protein" or "per nmol of P-450." Microsomes from the pyrazole-treated rats displayed an increase in binding spectrum with ethanol as the substrate as compared to controls, as well as type 2 binding spectrum with dimethyl sulfoxide and 2-butanol. These results suggest the possibility that pyrazole may induce an alcohol-preferring P-450 isozyme. By contrast, the 4-methylpyrazole treatment, besides increasing the oxidation of alcohols, also increased the oxidation of aminopyrine and the content of cytochrome P-450. The increase in the oxidation of alcohols and aminopyrine was primarily due to the increase in content of P-450 produced by the 4-methylpyrazole treatment. Binding spectra with dimethyl sulfoxide and 2-butanol were also observed after 4-methylpyrazole treatment; however, the 2-butanol-binding spectrum was a modified type 1 spectrum, not type 2.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of pyrazole and 4-methylpyrazole with hepatic microsomes: effect on cytochrome P-450 content, microsomal oxidation of alcohols, and binding spectra. 393 74

4-Methylpyrazole is a potent inhibitor of alcohol dehydrogenase and of ethanol metabolism. In vitro, 4-methylpyrazole was shown to inhibit microsomal oxidation of drugs and alcohols. Treatment of rats with 4-methylpyrazole at doses ranging from 0 to 300 mg per kg body wt per day for three days resulted in a dose-dependent increase in the content of liver microsomal cytochrome P-450. There was no change in the activity of NADPH-cytochrome P-450 reductase. 4-Methylpyrazole interacted with control microsomes to produce a type II binding spectrum, with a peak at 429 nm, and a trough at 392 nm. The magnitude of this spectral change was increased after 4-methylpyrazole treatment. Kinetic experiments indicated that the 4-methylpyrazole treatment lowered the dissociation constant (Ks) for 4-methylpyrazole. The maximal binding (Vs) was increased when expressed per mg microsomal protein, but not per nmol cytochrome P-450. Therefore, 4-methylpyrazole treatment can affect the microsomal mixed-function oxidase system in several ways, including binding to P-450 as well as inducing P-450.
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PMID:Increased content of cytochrome P-450 and 4-methylpyrazole binding spectrum after 4-methylpyrazole treatment. 397 3

Liver microsomes from rabbits treated chronically with ethanol were solubilized and fractionated to yield a new isozyme of cytochrome P-450 in a homogeneous state. This cytochrome, designated as isozyme 3a on the basis of its relative electrophoretic mobility, is distinct from the known terminal amino acid sequences. In addition, peptide mapping by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome has unusually high activity in the oxidation of ethanol and other alcohols to aldehydes and in the rho-hydroxylation of aniline as compared with the other isozymes of P-450. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH-cytochrome P-450 reductase and is stimulated by the presence of phosphatidylcholine, is not due to contamination by catalase or an NAD+-or NADP+-dependent alcohol dehydrogenase.
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PMID:Alcohol oxidation by isozyme 3a of liver microsomal cytochrome P-450. 668 96


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