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NADPH-cytochrome P450 reductase (CPR) is a key enzyme transferring electrons to cytochrome P450. In tomatoes (Solanum lycopersicum), two CPR genes, SlCPR1 and SlCPR2, were identified. In all the tested tomato tissues, SlCPR2 showed higher expression levels than SlCPR1. SlCPR2 expression increased significantly with jasmonic acid treatment. No significant changes were observed with salicylic acid or drought stress treatment. The cDNA of SlCPRs were expressed in Escherichia coli without any amino acid modification. And the heterologously expressed SlCPR enzymes were reacted with several protein and chemical substrates. SlCPR2 was more active than SlCPR1. Both SlCPR1 and SlCPR2 exhibited strong activity across a pH range of 6.0 to 9.0, with peak activity at pH 8.0. The study opens possibilities for CPR control, biocatalyst development, and exploring oxidase enzyme functions. [ABSTRACT FROM AUTHOR]

Abstract Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro , and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible. Highlights • Optimal conditions for expression of key enzymes in diosgenin synthesis in E. coli. • DzSE and DzCPR cooperate with each other to synthesis 2,3-oxidosqualene in vitro. • Endoplasmic reticulum serves as the active site for squalene oxidation. • Tissue expression showed the synthesis, transport and storage of 2,3-oxidosqualene. [ABSTRACT FROM AUTHOR]

Abstract Cytochrome P450 reductase (CPR) is the redox partner of various P450s involved in primary and secondary metabolism. Here, we identified and characterized two paralogs of cytochrome P450 reductase from Siraitia grosvenorii. There were two full-length CPR isoforms in the S. grosvenorii fruit transcriptome dataset. They had the same open reading frames of 2, 124 bp, encoding 707 amino acids. A phylogenetic analysis characterized both SgCPR1 and SgCPR2 as Class II dicotyledonous CPRs. The recombinant proteins SgCPR1 and SgCPR2 could reduce cytochrome c and ferricyanide in a NADPH-dependent manner. The SgCPR1 and SgCPR2 transcripts were detected in all examined tissues of S. grosvenorii , and in fresh fruit, they had expression patterns similar to several key enzymes that require CPR as a partner during their biosynthesis. The expression levels of the SgCPR s were induced after a methyl jasmonate treatment. The extracts from yeast co-expressing SgCPR1/SgCPR2 and the cytochrome P450 enzyme CYP76AH1 produced ferruginol, indicating the positive effects of SgCPR1/SgCPR2 on the CYP76AH1 activity. A docking analysis confirmed the experimentally deduced functional activities of SgCPR1 and SgCPR2 for NADPH, FAD and FMN. Thus, SgCRP1 and SgCPR2 are both likely to participate in secondary metabolism, especially mogroside biosynthesis in S. grosvenorii. [ABSTRACT FROM AUTHOR]

NADPH-cytochrome P450 reductase (CPR) plays an essential role in the cytochrome P450 enzyme system, which aids in the metabolism of endogenous and exogenous compounds including the detoxification of insecticides. In this study, the CPR transcript in Aphis (Toxoptera) citricidus (Kirkaldy) was cloned, and the deduced amino acid sequence contained an N-terminal membrane anchor, three conserved binding domains (flavin mononucleotide, flavin adeninedinucleotide, and nicotinamide adenine dinucleotide phosphate), a flavin adeninedinucleotide-binding motif, and catalytic residues. Based on phylogenetic analysis, AcCPR was grouped in the hemipteran branch. AcCPR was ubiquitously expressed at all developmental stages and was most abundant in the adults and least abundant in third instar nymphs. Compared with other tested tissues of adults, the expression level of AcCPR was significantly high in the gut. Feeding double-stranded RNA of AcCPR reduced the AcCPR mRNA level and the activity of AcCPR in aphids, and the treated insects exhibited higher susceptibility to abamectin than the control group. Furthermore, the heterologous overexpression of AcCPR in Sf9 cells resulted in a greater viability than control cells when treated with abamectin. All results demonstrated that AcCPR may contribute to the resistance of A. citricidus to abamectin, and CPR may be a potential target for novel insecticide design or a new factor in the development of insecticide resistance. [ABSTRACT FROM AUTHOR]

Background NADPH-cytochrome P450 reductase (CPR) plays important roles in cytochrome P450-mediated metabolism of endogenous and exogenous compounds, and participates in cytochrome P450-related detoxification of insecticides. However, the CPR from Locusta migratoria has not been well characterized and its function is still undescribed. Results The full-length of CPR gene from Locusta migratoria ( LmCPR ) was cloned by RT-PCR based on transcriptome information. The membrane anchor region, and 3 conserved domains (FMN binding domain, connecting domain, FAD/NADPH binding domain) were analyzed by bioinformatics analysis. Phylogenetic analysis showed that LmCPR was grouped in the Orthoptera branch and was more closely related to the CPRs from hemimetabolous insects. The LmCPR gene was ubiquitously expressed at all developmental stages and was the most abundant in the fourth-instar nymphs and the lowest in the egg stage. Tissue-specific expression analysis showed that LmCPR was higher expressed in ovary, hindgut, and integument. The CPR activity was relatively higher in Malpighian tubules and integument. Silencing of LmCPR obviously reduced the enzymatic activity of LmCPR, and enhanced the susceptibility of Locusta migratoria to carbaryl. Conclusion These results suggest that LmCPR contributes to the susceptibility of L. migratoria to carbaryl and could be considered as a novel target for pest control. [ABSTRACT FROM AUTHOR]

NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus , a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR ( LsCPR ) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92 kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera , Nilaparvata lugens , Cimex lectularius and Anopheles gambiae , respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus , as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200 μg/mL of LsCPR double-stranded RNA (ds LsCPR ) by the planthopper for 5 days significantly reduced the transcription level of LsCPR . Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a susceptible (YN) strain. These data further suggested that the P450-mediated metabolic detoxification of xenobiotics might be an important mechanism for buprofezin resistance in L. striatellus . [ABSTRACT FROM AUTHOR]

While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H - -TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. [ABSTRACT FROM AUTHOR]

NADPH-cytochrome P450 reductase was purified from Lake Van fish liver microsomes by primary and secondary DEAE-cellulose column chromatograph with 20.46 μM/min/mg enzyme specific activities, 54.4% purification yield, and purification of 38-fold. The purity of the enzyme was established, and its monomer molecular weight was determined by SDS-polyacrylamide gel electrophoresis. SDS-PAGE results showed a single band and the molecular weight of NADPH-cytochrome P450 reductase was 70 kDa. In addition, optimum ionic strength, optimum pH, optimum temperature, and stable pH values were determined for the enzyme in the kinetic studies performed. KM and Vmax were determined for NADPH and cytochrome c. Effects of some metals ions, antibiotics, and some other drugs used in aquarium fisheries on the activity of the enzyme were investigated. IC50 values and Ki values of metals showing an inhibitory effect were calculated. [ABSTRACT FROM AUTHOR]

The cDNAs for cytochrome P450 monooxygenase (designated as CYP97C27 by D. Nelson's group) and NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from Croton stellatopilosus leaves, which actively biosynthesise plaunotol (18-OH geranylgeraniol). CYP97C27 and CPR I contain open reading frames encoding proteins of 471 and 711 amino acids with predicted molecular masses of 53 and 79 kDa, respectively. By aligning the deduced sequences of CYP97C27 and CPR I with other plant species, all functional domains of CYP97C27 (heme and oxygen binding) and CPR I (CYP- and FMN, FAD, and NADPH cofactor binding) were identified. Amino acid sequence comparison indicated that both CYP97C27 (85–93%) and CPR I (79–83%) share high sequence identities with homologous proteins in other plant species, suggesting that CYP97C27 belongs to the CYP97 C subfamily and that CPR I belongs to class I of the dicotyledonous CPR. Functional characterisation of both enzymes, produced in Escherichia coli (pET32a/BL21(DE3)) as recombinant proteins, showed that simultaneous incubation of CYP97C27 and CPR I with the substrate geranylgeraniol (GGOH) and coenzyme NADPH led to formation of the product plaunotol. In C. stellatopilosus , the levels of the CYP97C27 and CPR I transcripts were highly correlated with those of several mRNAs involved in the plaunotol biosynthetic pathway, suggesting that CYP97C27 and CPR I are the enzymes that catalyse the last hydroxylation step of the pathway. [ABSTRACT FROM AUTHOR]

Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH-cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V and K of PkCPR were found to be 5.8 ± 0.05 μmol min mg, 8.1 ± 0.12 μmol min mg and 7.8 μM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa. [ABSTRACT FROM AUTHOR]

Abstract: CYP3A4 is the most abundant cytochrome P450 in the human liver. The expression level of CYP3A4 when coexpressed with cytochrome b 5 (cyt b 5) in Escherichia coli was 20–60% higher than that when it was expressed alone over an extended period (48–72h). This time-dependent elevation in coexpression with cyt b 5 was a result of an increase in CYP3A4 mRNA half-life; no significant change in CYP3A4 degradation was seen in the bacterial protease fraction. These results suggest that the higher CYP3A4 levels observed upon coexpression with cyt b 5 primarily resulted from CYP3A4 mRNA stabilization by cyt b 5. [Copyright &y& Elsevier]

The aim of the present study was to test a new hypothesis that brain cytochrome P450 reductase (CPR) and CPR-dependent enzymes play important roles in behavioral performance. A mouse model with brain neuron-specific deletion of the Cpr gene (brain- Cpr -null) was recently generated. Brain- Cpr -null mice and wild-type (WT) littermates were compared in a variety of behavioral assays. Notable differences were found in the exploratory behavior assay: for both males and females, activity in the center of the chamber was significantly higher for brain- Cpr -null than for WT mice on days 2 and 3 of the assay, although no significant difference was found between the two groups in anxiety-like behavior in the elevated zero maze. Furthermore, in the fear-conditioning assay, brain- Cpr -null mice exhibited significantly less activity suppression than did WT controls. This deficit in activity suppression was not accompanied by any difference between WT and brain- Cpr -null mice in nociceptive responses to foot shocks. Abnormal activity suppression was also observed in both male and female brain- Cpr -null mice during the contextual memory test. However, in the Morris water maze assay, the brain- Cpr -null and WT mice were indistinguishable, indicating normal spatial memory in the mutant mice. These data collectively indicate a novel role of the Cpr gene in fear conditioning and memory. [ABSTRACT FROM AUTHOR]

Abstract: 7-Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis. The enzyme utilizes NADPH as a source of electrons and has been reported to require NADPH-cytochrome P450 reductase (POR) as its redox partner. To test this hypothesis, microsomes were prepared from the livers of mice in which hepatic cytochrome P450 reductase expression was extinguished during maturation. These microsomes contained negligible levels of POR but had 2.5-fold greater DHCR7 activity than did microsomes from wild-type mice. Consistent with this greater activity, immunoblot analysis of DHCR7 expression indicated that DHCR7 protein levels were elevated 2-fold in POR-null microsomes. Addition of POR to these microsomes provided no stimulation of DHCR7 activity, confirming the lack of a role for POR in DHCR7 activity. Because the original observation that POR was necessary for DHCR7 activity was based, in part, on antibody inhibition studies with POR antibody, the ability of an antibody to the full-length POR protein to inhibit DHCR7 activity and cytochrome c reductase activity was tested; the antibody had no effect on DHCR7 activity but decreased cytochrome c reductase activity (a POR-catalyzed reaction) by 50%. Immunoblot analysis further demonstrated no cross-reactivity between POR and DHCR7 with antibodies to either protein. We conclude that cytochrome P450 reductase is not involved in 7-dehydrocholesterol reductase activity. [Copyright &y& Elsevier]

Abstract: Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, carbon monoxide (CO) and ferrous iron via three successive monooxygenase reactions, using electrons provided by NADPH-cytochrome P450 reductase (CPR) and oxygen molecules. For cleavage of the oxaporphyrin ring of ferrous α-verdoheme, an intermediate in the HO reaction, involvement of a verdoheme π-neutral radical has been proposed. To explore this hypothetical mechanism, we performed electrochemical reduction of ferrous α-verdoheme–rat HO-1 complex under anaerobic conditions. Upon binding of CO, an O2 surrogate, the midpoint potential for one-electron reduction of the oxaporphyrin ring of ferrous α-verdoheme was increased from −0.465 to −0.392V vs the normal hydrogen electrode. Because the latter potential is close to that of the semiquinone/reduced redox couple of FAD in CPR, the one-electron reduction of the oxaporphyrin ring of CO-bound verdoheme complexed with HO-1 is considered to be a thermodynamically likely process. Indeed the one-electron reduced species, [FeII(verdoheme•)], was observed spectroscopically in the presence of CO in both NADPH/wild-type and FMN-depleted CPR systems under anaerobic conditions. Under physiological conditions, therefore, it is possible that O2 initially binds to the ferrous iron of α-verdoheme in its complex with HO-1 and an electron is subsequently transferred from CPR, probably via FAD, to the oxaporphyrin ring. [ABSTRACT FROM AUTHOR]

Abstract: The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species. [Copyright &y& Elsevier]

Abstract: The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9±0.5 and 2.6±0.4 nmol/min/mg protein (mean±SD, n =3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61±6.01 µM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species. [Copyright &y& Elsevier]

Mammalian NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 enzymes and other several microsomal enzymes. It also catalyzes the one-electron reduction of many chemicals and drugs. Reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by CPR was assessed as a method for monitoring CPR activity. The electrons released from NADPH by CPR were transferred to CTC in the reaction medium, and CTC reduction activity could be assessed spectrophotometrically and spectrofluorometrically. The reduction kinetics of CTC follows classical Michaelis–Menten kinetics ( K m = 50 μM, k cat = 2,520 min−1). This method offers a continuous assay of the enzymatic activity of CPR. [ABSTRACT FROM AUTHOR]

Abstract: The technology for over-expressing NADPH-cytochrome P450 reductase (CPR), a diflavin-containing enzyme, offers the opportunity to develop enzymatic systems for environmental detoxication and bioconversions of drugs, pesticides and fine chemicals. In this study, Bacillus subtilis was chosen to express rat CPR (rCPR) because of its capacities for high protein production and spore formation. rCPR was expressed in B. subtilis DB104 under the transcriptional control of an IPTG-inducible fusion promoter of P groE and P tac . The expressed rCPR was released into the culture medium after sporulation by autolysis of the host cell. It was associated with and displayed on the spore surfaces; this was confirmed by measuring rCPR activity in purified spores and analyzing its accessibility to anti-rCPR antibodies using flow cytometry. The spore-displayed rCPR was able to reduce cytochrome c and ferricyanide, and also assisted in the O-deethylation of 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin (EFC) by human cytochrome P450 1A2, indicating that it was functionally active. Spore surface display of rCPR in B. subtilis appears to be useful for preparing cytochrome P450-related enzymes, and spore biocatalysts of rCPR are likely to have wide biotechnological applications. [Copyright &y& Elsevier]

Abstract: Information on the mechanism of metal ion inhibition of NADPH-cytochrome P450 reductase is limited. The purpose of the present paper was to elucidate in vitro effect of Hg+2, Cd+2, Ni+2, Cr+3 and Zn+2 ions on the purified mullet NADPH-cytochrome P450 reductase. NADPH-cytochrome P450 reductase was purified from detergent-solubilized liver microsomes from leaping mullet (Liza saliens). All of the metal ions caused inhibition of the enzyme activity except Zn+2. At 50μM metal concentration, Hg+2 inhibited the cytochrome P450 reductase activity completely (100%), while, at the same concentrations, Cd+2, Cr+3 and Ni+2 caused 66%, 65% and 37% inhibition, respectively. At 50μM metal concentration, Zn+2 had no apparent effect on cytochrome P450 reductase activity. The IC50 values of HgCl2, CrCl3, CdCl2 and NiCl2 were estimated to be 0.07μM, 24μM, 33μM and 143μM, respectively. Of the metal ions tested, Hg+2 exhibited much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than the others. All four metal ions displayed noncompetitive type of inhibition mechanism for the purified reductase as analyzed by Dixon plot. K i values of Hg+2, Cr+3, Cd+2, and Ni+2 were calculated from Dixon plots as 0.048μM, 18μM, 73μM and 329μM, respectively. [Copyright &y& Elsevier]

Abstract: Studies with repair-deficient mice and other experiments suggest that oxidative DNA modifications are generated in all types of cells even under physiological conditions and that this type of endogenous DNA damage contributes to spontaneous cancer incidence. However, the cellular sources of reactive oxygen species that are relevant for nuclear oxidative DNA damage are largely unknown. Here, we report that expression of human NADPH-cytochrome P450 reductase (hOR) in cultured V79 Chinese hamster cells gives rise to elevated basal levels of oxidative purine modifications after depletion of glutathione. Also, the basal levels of micronuclei are increased in the hOR-expressing cells, and again the effect is enhanced when the antioxidant defense system of the cells is diminished by depletion of glutathione. The oxidative DNA damage is increased when duroquinone, a substrate of hOR, is added, both in the presence and absence of glutathione. In contrast, hOR-expressing cells are similarly sensitive as the parental cells when oxidative DNA damage and micronuclei are induced by a mechanism independent of hOR, i.e., exposure to bromate. The results identify hOR as a potential source of endogenous oxidative DNA damage and subsequent genetic instability in mammalian cells. [Copyright &y& Elsevier]

The unifying thread of this review involves NADPH-cytochrome P450 reductase (CYPOR), the microsomal enzyme responsible for transferring electrons to cytochromes P450, as well as several other monooxygenase systems, a lifelong interest of the corresponding author. The intersection of her research with that of Dr. David Kupfer, their resulting collaboration, and the beginning of a long-standing study of fatty acid- and eicosanoid-metabolizing cytochromes P450 (CYP4A gene subfamily), including the role of cytochrome b 5, will be reported. The culmination of this interest now involves purification and characterization of the human mutants of CYPOR that have been implicated in pathologies, such as Antley-Bixler syndrome. [ABSTRACT FROM AUTHOR]

Abstract: NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD–FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH /FMNH2 couple donates electrons to cytochrome P450 at constant oxidation–reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed. [Copyright &y& Elsevier]

NADPH-cytochrome P450 reductase (CPR) is a membrane-bound flavoprotein that interacts with the membrane via its N-terminal hydrophobic sequence (residues 1-56). CPR is the main electron transfer component of hydroxylation reactions catalyzed by microsomal cytochrome P450s. The membrane-bound hydrophobic domain of NADPH-cytochrome P450 reductase is easily removed during limited proteolysis and is the subject of spontaneous digestion of membrane-binding fragment at the site Lys56-Ile57 by intracellular trypsin-like proteases that makes the flavoprotein very unstable during purification or expression inE. coli. The removal of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase results in loss of the ability of the flavoprotein to interact and transfer electrons to cytochrome P450. In the present work, by replacement of the lysine residue (Lys56) with Gln using site directed mutagenesis, we prepared the full-length flavoprotein mutant Lys56Gln stable to spontaneous proteolysis but possessing spectral and catalytic properties of the wild type flavoprotein. Limited proteolysis with trypsin and protease fromStaphylococcus aureusof highly purified and membrane-bound Lys56Gln mutant of the flavoprotein as well as wild type NADPH-cytochrome P450 reductase allowed localization of some amino acids of the linker fragment of NADPH-cytochrome P450 reductase relative to the membrane. During prolong incubation or with increased trypsin ratio, the mutant form showed an alternative limited proteolysis pattern, indicating the partial accessibility of another site. Nevertheless, the membrane-bound mutant form is stable to trypsinolysis. Truncated forms of the flavoprotein (residues 46-676 of the mutant or 57-676 of wild type NADPH-cytochrome P450 reductase) are unable to transfer electrons to cytochrome P450c17 or P4503A4, confirming the importance of the N-terminal sequence for catalysis. Based on the results obtained in the present work, we suggest a scheme of structural topology of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase in the membrane. [ABSTRACT FROM AUTHOR]

Recently we have shown that ferric α-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem. 274 , 18196–18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al . have reported that the major reaction product of ferric α-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous α-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta 1432 , 203–213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. α-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of α-hydroxyhaem with haem oxygenase may be small and a substantial amount of free α-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2 . Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase. [ABSTRACT FROM AUTHOR]

Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11α position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78 kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78 kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11α-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c3+. Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction. [Copyright &y& Elsevier]

We examined the paraquat detoxicative system in mouse livers. The survival rate of mice receiving 50 mg/kg paraquat was 41% at 7 days and significantly rose to 88, 64, 69% with pretreatment with phenytoin, phenobarbital, and rifampicin, respectively. Phenytoin induced activity in NADPH-cytochrome P450 reductase, CYP3A, CYP2B, and CYP2C that was 3 to 4 times higher than that of the controls. Phenobarbital induced CYP2B and rifampicin induced CYP3A, respectively, in addition to NADPH-cytochrome P450 reductase. 3-Methylcholanthrene did not induce these enzymes and did not alter the survival rate. All the mice pretreated with CoCl2 (a CYP synthesis inhibitor) or SKF 525-A (a CYP inhibitor) were dead after 5 days, and troleandomycin (a CYP3A-specific inhibitor) also reduced the survival rate. When cell homogenates were incubated with paraquat and NADPH, paraquat decreased and its metabolic intermediate paraquat-monopyridone was formed. Troleandomycin inhibited the decrease in paraquat and increased the monopyridone. After making a subfraction of the homogenate, monopyridone was produced in the postmicrosomal 105,000g supernatant, but not in the microsomes. The pretreatment of mice with phenytoin decreased the monopyridone in the postmitochondrial fraction, but did not affect the supernatant. These results indicated that paraquat was first metabolized in the postmicrosomal supernatant into monopyridone, and that may have been subsequently hydroxylated by the microsomes. Repeated intravenous injections of α-tocopherol to paraquat-loaded mice significantly reduced the paraquat mortality and when these mice were pretreated with rifampicin, 100% of them survived. These studies demonstrate that postmitochondrial fractions play an important role in paraquat detoxication metabolism, and that the combination of CYP induction and α-tocopherol administration is highly useful for the survival of paraquat-exposed mice. [Copyright &y& Elsevier]

We reported previously that various naphthoquinone derivatives inhibited cytochrome P450-dependent monooxygenase of liver and placenta microsomes [Muto, N. et al. (1987) Biochem. Biophys. Res. Commun. 146, 487–494]. To understand the complex inhibitory behaviors that were observed, it is desirable to study the relationship between structure and inhibitory activity of naphthoquinones in a simplified system containing a single P450 species. In the present study, the inhibitory effects of six derivatives of 1,4-naphthoquinone (hereafter referred to as NQ) on rat cytochrome P4501Al-dependent 7-ethoxycoumarin O-deethylation were examined using yeast microsomes containing overexpressed rat P4501A1. Of these, 2-methyl-5-hydroxy-NQ, 2-methyl-NQ, 2-hydroxy-NQ, and NQ showed competitive inhibition, whereas 5,8-dihydroxy-NQ and 5-hydroxy-NQ showed noncompetitive inhibition. Judging from the inhibitor constant (K1), the binding affinity of the four competitive inhibitors for the substrate-binding pocket of P4501A1 is in the order: 2-CH3-5-OH-NQ > 2-CH3-NQ > NQ > 2-OH-NQ. On binding with P4501A1, 2-CH3-5-OH-NQ, 2-CH3-NQ, and NQ induced distinct Type II, Type I, and reverse Type I spectra, respectively. These results indicate that methyl and hydroxyl groups introduced into NQ have unique effects on their binding mode and binding affinity. [ABSTRACT FROM AUTHOR]

Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology. [ABSTRACT FROM AUTHOR]

We have purified membrane-bound fatty acid (ω-1-ω-3) hydroxylase of the fungus Fusarium oxysporum MT-811 and found that the activity depends on a single polypeptide with an apparent Mr value of 118,000. The purified hydroxylase exhibited spectral characteristics of cytochrome P450 (P450), and could catalyze the hydroxylation without the aid of any other proteinaceous components, such as NADPH-P450 reductase. These properties of the fungal hydroxylase are the same as those of bacterial P450BM3 of Bacillus megaterium, a catalytically self-sufficient fused protein of P450 and its reductase. Other properties of the two enzymes, such as molecular weight, high catalytic turnover, and the regiospecificity of the hydroxylating position, were also almost identical. Further, the fungal hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase structurally and functionally bears a close resemblance to P450BM3, although it is membrane-bound, unlike the bacterial counterpart.On the other hand,a unique phenomenon was found with the fungal hydroxylase: its NADPH-cyto-chrome c- or NADPH-menadione reductase activity was enhanced enormously upon binding of its substrate (fatty acid). This appears to be the first instance in which the reactivity of P450 reductase against an artificial electron acceptor was enhanced by the binding of the substrate (to be hydroxylated) to P450. These results raise interesting questions about the molecular evolution of P450. Here we term the fungal hydroxylase cytochrome P450foxy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To analyse the structure and function of NADPH-cytochrome p450 reductase (CYPOR or CPR) from Plasmodium falciparum (Pf), and to predict its'' drug target and vaccine target. Methods: The structure, function, drug target and vaccine target of CPR from Plasmodium falciparum were analyzed and predicted by bioinformatics methods. Results: PfCPR, which was older CPR, had close relationship with the CPR from other Plasmodium species, but it was distant from its hosts, such as Homo sapiens and Anopheles. PfCPR was located in the cellular nucleus of Plasmodium falciparum. 335aa–352aa and 591aa – 608aa were inserted the interior side of the nuclear membrane, while 151aa–265aa was located in the nucleolus organizer regions. PfCPR had 40 function sites and 44 protein–protein binding sites in amino acid sequence. The teriary structure of 1aa–700aa was forcep–shaped with wings. 15 segments of PfCPR had no homology with Homo sapien CPR and most were exposed on the surface of the protein. These segments had 25 protein–protein binding sites. While 13 other segments all possessed function sites. Conclusions: The evolution or genesis of Plasmodium falciparum is earlier than those of Homo sapiens. PfCPR is a possible resistance site of antimalarial drug and may involve immune evasion, which is associated with parasite of sporozoite in hepatocytes. PfCPR is unsuitable as vaccine target, but it has at least 13 ideal drug targets. [Copyright &y& Elsevier]

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR's degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP's substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound. [ABSTRACT FROM AUTHOR]