NADPH-cytochrome reductase

In a recent investigation to develop novel cytochrome P450 biocatalysts, DNA shuffling was used to produce chimeric cytochrome P450s mutants with enhanced biocatalytic activities, which were then co-expressed with NADPH-cytochrome reductase in E. coli to form an efficient system, in this case demonstrated to be effective for indole oxidation [69]. 

KC1 gradient (right ordinate). In these fractions, neither NADPH cytochrome reductase activity, nor cytochrome bs could be detected. From Balny et al. (1975). Reprinted with permission of Analytical Biochemistry. Copyright by Academic Press. 

Glutamate dehydrogenase and pyruvate dehydrogenase. Glucose 6-phosphatase NADPH-cytochrome reductase Fructose diphosphate aldolase Galactosyltransferase... 

VO (a) Yeast NADH (NADPH) Cytochrome reductase Cytochrome Oleoyl-CoAor oleoyl-PL... 

Microsomal cytochromes P450 are membrane-bound. They accept electrons from a microsomal NADPH-cytochrome reductase, containing flavin adenine dinucleotide and flavin mononucleotide. All cytochromes P450 metaboUsing drugs and xeno-biotica isolated so far belong to this class. 

He/minthosporium (15). The mode of action is considered to be inhibition of the enzyme NADPH-cytochrome C reductase, which results in the generation of free radicals and/or peroxide derivatives of flavin which oxidize adjacent unsaturated fatty acids to dismpt membrane integrity (16) (see Enzyme inhibitors). 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

To summarize, squalene epoxidase is a flavoprotein capable of catalyzing the insertion of oxygen into the 2,3-double bond of squalene to give 2,3-oxidosqualene, with the second oxygen atom from 02 being reduced to water. The reducing equivalents necessary for this transformation are relayed from NADPH through NADPH-cytochrome c reductase to the flavin cofactor of the epoxidase. 

Asymmetric oxidation of this sulphide was also catalyzed by two isocytochromes P 450 purified from phenobarbital induced rat liver309. Both P 450 isocytochromes, termed PB-1 and PB-4, when reconstituted with purified rat liver NADPH-cytochrome P 450 reductase and cytochrome b5 afforded ethyl p-tolyl sulphoxide with S-configuration at the sulphur atom. In the case of PB-1 optical purity of this sulphoxide was 58% whereas with PB-4 it was 78%. 

The microsomal fraction consists mainly of vesicles (microsomes) derived from the endoplasmic reticulum (smooth and rough). It contains cytochrome P450 and NADPH/cytochrome P450 reductase (collectively the microsomal monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases, glucuronyl transferases, and other enzymes that metabolize xenobiotics. The 105,000 g supernatant contains soluble enzymes such as glutathione-5-trans-ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant contains all of the types of enzyme listed earlier. 

Cytochrome P450s catalyze reactions that introduce one atom of oxygen derived from molecular oxygen into the substrate, yielding a hydroxylated product. NADPH and NADPH-cytochrome P450 reductase are involved in the complex reaction mechanism. 

DMN oxidative demethylation has been shown to be a liver mi-crosome cytochrome P-450 monooxygenase (10) Lotlikar et al. (11) found that a reconstituted enzyme system, consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and phosphatidyl choline was effective in catalyzing the demethylation of DMN. The most commonly accepted mechanism for the oxidative demethylation of DMN and, by extension, of other dialkyInltrosamlnes is shown in Scheme 1. 

Sasame, H.A. Ames, M.M. and Nelson, S.D. Cytochrome P-450 and NADPH cytochrome c reductase in rat brain Formation of catechols and reactive catechol metalbolites. Biochem Biophys Res Commun 78 919-926, 1977. 

Vail, R.B., Homann, M.J., Hanna, I. and Zaks, A. (2005) Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH P450 reductase expressed in Escherichia coli. Journal of Industrial Microbiology Biotechnology, 32, 67-74. 

Yamazaki, H., Ueng, Y.F., Shimada, T. and Guengerich, F.P. (1995) Roles of divalent metal ions in oxidations catalyzed by recombinant cytochrome P450 3A4 and replacement of NADPH-cytochrome P450 reductase with other flavoproteins, ferredoxin, and oxygen surrogates. Biochemistry, 34, 8380—8389. 

E. G., Creation of polarized cells coexpressing CYP3A4, NADPH cytochrome P450 reductase and MDRl/P-glycoprotein, Pharm. Res. 2000, 37, 803-810. 

Saito et al. (134) found that the cytosolic nitroreductase activity was due to DT-diaphorase, aldehyde oxidase, xanthine oxidase plus other unidentified nitroreductases. As anticipated, the microsomal reduction of 1-nitropyrene was inhibited by 0 and stimulated by FMN which was attributed to this cofactor acting as an electron shuttle between NADPH-cytochrome P-450 reductase and cytochrome P-450. Carbon monoxide and type II cytochrome P-450 inhibitors decreased the rate of nitroreduction which was consistent with the involvement of cytochrome P-450. Induction of cytochromes P-450 increased rates of 1-aminopyrene formation and nitroreduction was demonstrated in a reconstituted cytochrome P-450 system, with isozyme P-448-IId catalyzing the reduction most efficiently. 

Microsomal NADPH-Cytochrome P-450 Reductase and NADH cytochrome b5 Reductase... 

MICROSOMAL NADPH-CYTOCHROME P-450 REDUCTASE AND NADH CYTOCHROME b5 REDUCTASE... 

The primary function of flavoprotein NADPH-cytochrome P-450 reductase is the hydro-xylation of various substrates, which occurs during electron transfer from NADPH to cytochrome P-450 [1] ... 

While cytochrome P-450 catalyzes the interaction with substrates, a final step of microsomal enzymatic system, flavoprotein NADPH-cytochrome P-450 reductase catalyzes the electron transfer from NADPH to cytochrome P-450. As is seen from Reaction (1), this enzyme contains one molecule of each of FMN and FAD. It has been suggested [4] that these flavins play different roles in catalysis FAD reacts with NADPH while FMN mediates electron... 

It has been believed that P-450 reduction by NADPH cytochrome P-450 reductase is a biphasic process, but it was recently shown [7] that some P-450 cytochromes are reduced with single-exponential kinetics and that the presence of substrate is not an obligatory condition for the reduction of all P-450 forms. Thus, the kinetics of reduction of various ferric P-450 cytochromes possibly depends on many factors such as substrate, rate-limiting step, etc. 

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