NADPH-P450 reductase

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. 

This electron transfer pathway may partially explain the extraordinarily high efficacy of nitroprusside as it provides single electrons at a sufficiently negative potential to reduce both P450 (E° = — 0.17 to — 0.35 V) [148] and nitroprusside. Since NADPH/P450 reductase has been shown to be an efficient activator of this drug [43], NO may be evolved by NO-synthase reduction of nitroprusside at the same sites where NO is produced physiologically. 

Sakaki,T., Kominami, S., Takemori, S., Ohkawa, H., Akiyoshi-Shibata, M. Yabusaki, Y. (1994). Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase. Biochemistry, 33, 4933-9. 

NADPH-P450 Reductase" 1 NADPH-P450 Reductase"... 

Hakamatsuka, T., Hashim, M.E., Ebizuka, Y. and Sankawa, U. (1991) P450-dependent oxidative rearrangement in isoflavone biosynthesis reconstitution of P450 and NADPH P450 reductase. Tetrahedron, 47, 5969-78. 

Wolf, C.R., Moll, E., Friedberg, T., Oesch, F., Buchmann, A., Knhl-mann, W.D. Kunz, H.W. Characterization, localization and regulation of a novel phenobarbital-inducible form of cytochrome P 450, compared with three further P450-isoenzymes, NADPH P450-reductase glutathione transferases and microsomal epoxide hydrolase. Carcinogenesis 1984 5 993-1001... 

Reduction Cytochrome P450 NADPH-P450 reductase Carbonyl reductase... 

Under aerobic and anaerobic conditions, several reduction reactions can be catalyzed by the intact P450 monooxygenase system or only by its flavopro-tein component, NADPH-P450 reductase. 

Aryal, P. et al., Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase, Mutat. Res., 442, 113, 1999. 

System of microsomal MO comprises three main catalytic components. Cytochrome P450 system (reduced flavoprotein oxygen oxidoreductase, EC 1.14.14.1) participate in hydroxylation reactions through processes of oxidoreduction. In oxidative reactions very important is the presence of oxygen, while in reduction reactions electrons are transferred from heme. In some of these reactions enzyme NADPH-P450 reductase (NADPH fcrricytochromc oxidoreduc-... 

Current bacterial strains expressing single CYPs together with human NADPH P450 reductase are listed in Tab. 3.1. In addition, strains expressing the following complementary enzymes are available human NADPH P450 reductase, human cytochrome hs and human NADPH cytochrome bs reductase. 

Notice that a prerequisite for substrate binding (step 1) is that the heme iron of cytochrome P450 must be at the +3 valency (ferric iron). Subsequent to binding an electron is transferred to P450 (step 2) via NADPH -P450 reductase (or POR as shovm in Figure 8.5). This reduces the heme iron to ferrous iron. 

NADPH-P450 reductase NADPH-P450 reductase ... 

Rodrigues, A.D. Mulford, D.J. Lee, R.D. Surber, B.W. Kukulka, M.J. Ferrero, J.L. Thomas, S.B. Shet, M.S. Estabrook, R.W. In vitro metabolism of terfenadine by a purified recombinant fusion protein containing cytochrome P4503A4 and NADPH-P450 reductase. Comparison to human liver microsomes and precision-cut liver tissue slices. Drug Metab.Dispos., 1995, 23, 765-775... 

Ram, P.A. and D.J. Waxman (1992). Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms. J. Biol. Chem. 267, 3294-3301. 

Fisher, C.W., M.S. Shet, D.L. Caudle, C.A. Martin-Wixtrom, and R.W. Estabrook (1992). High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein. Proc. Natl. Acad. Sci. USA 89, 10817-10821. 

Shet, M., C.W. Fisher, P.L. Holmans, and R.W. Estabrook (1996). Xhe omega-hydroxlyation of lau-ric acid Oxidation of 12-hydroxylauric acid to dodecanedioic acid by a purified reeombinant fusion protein containing P450 4A1 and NADPH-P450 reductase. Arch. Biochem. Biophys. 330,199-208. 

Helvig, C. and J.H. Capdevila (2000). Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains. Biochemistry 39, 5196-5205. 

Hayashi, K., X. Sakaki, S. Kominami, K. Inouye, and Y. Yabusaki (2000). Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome bS in yeast. Arch. Biochem. Biophys. 381, 164—170. 

K. Ohyama, N. Hatanaka, S. Asahi et al. (2002). Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Protein Expr. Purif. 24, 329-337. 

In early work in this field, this point would have been the demonstration of the reaction of interest with an enzyme purified from tissue. Today P450 proteins are generally produced in recombinant systems and seldom purified from tissue sources. In routine practice in the pharmaceutical industry, new reactions are examined with a battery of the major recombinant human (liver) P450s, many of which are available from commercial sources. Systems used for expression include bacteria, yeast, baculovirus (-infected insect cells), and mammalian cells. The P450s need not be purified for these comparisons hut must have suitable provision for NADPH-P450 reductase in a crude system (and cytochrome... 

Modi et reported differences in product profiles of P450 2D6 reactions supported with artificial oxygene surrogates and NADPH-P450 reductase, and interpreted these as evidence for an allosteric influence of the reductase. Subsequent experiments in this laboratory did not support this conclusion and are in accord with some differences in the chemical mechanisms for the oxygen surrogates. ... 

The interesting observation was made that the LI3IF mutant catalyzes only to-l hydroxylation and not co-hydroxylation of lauric acid . Residue 131 also controlled access to substituted imidazole inhibitors. Interestingly, some of the results on binding of imidazoles provide evidence that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of both NADPH-P450 reductase and... 

Imaoka et found that functional P450 4B1 could be successfully expressed as a fusion protein with NADPH-P450 reductase. They were also successful in developing transgenic mice in which functional P450 was expressed in liver. The authors postulate that expression in the presence of auxiliary proteins (NADPH-P450 reductase, bj) may stabilize P450 4B1 (ref [972]). With these systems, it was possible to demonstrate that... 

Relatively little is known about the active site of P450 5A1 beyond the information about the reactions presented above. As indicated, the protein does not bind NADPH-P450 reductase. Presumably, the active site is rather specific, although iodosylbenzene could be utilized as an oxygen surrogate. 

P450 llBl is synthesized in the cytosol and directed to the mitochondria with a 24-residue N-terminal targeting sequence (where this is lost after entry). As with the other four (exclusively) mitochondrial P450s (Table 10.1 and Figure 10.14), P450 IIBI receives electrons fi-om adrenodoxin instead of NADPH-P450 reductase. 

F.P. Guengerich, and K. Inoue (2000). Roles of NADPH-P450 reductase in the 0-deethylation of... 

FIGURE 2.8 Abortive redox cycling and oxidation of NADPH in a system involving a heterocyclic arylamine (Kim et al., 2004). NPR = NADPH-P450 reductase. 

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