Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

Djordjević, Snežana; Roberts, David L.; Wang, M.; Shea, Thomas M.; Camitta, M; Masters, Bettie Sue Siler; Kim, J. J P

doi:10.1073/pnas.92.8.3214

Cited by 31 publications

(20 citation statements)

References 44 publications

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“…Our studies also demonstrated that redox cycling by recombinant cytochrome P450 reductase and rat liver microsomes was suppressed by DPI, an inhibitor of flavoproteins [20, 34]. These findings are consistent with the fact that cytochrome P450 reductase contains both FAD and FMN [28]. Of interest was our observation that diquat was more active than paraquat in generating hydrogen peroxide and hydroxyl radicals during redox cycling, a finding likely due to the more positive redox potential of diquat (-350 mV) when compared to paraquat (-446 mV) [6, 35].…”

Section: Discussionsupporting

confidence: 85%

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Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase

Fussell

Udasin

Gray

et al. 2011

Free Radical Biology and Medicine

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Diquat and paraquat are non-specific defoliants that induce toxicity in many organs including the lung, liver, kidney and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In the present studies, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese Hamster Ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective in generating ROS when compared to paraquat (KM = 1.0 and 44.2 μM, respectively for H2O2 generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5 μM, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (Vmax ≈ 6.0 nmoles H2O2/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity.

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Section: Discussionsupporting

confidence: 85%

“…2, panels E and F). These inhibitory effects of DPI are consistent with the fact that cytochrome P450 reductase and other enzymes known to mediate redox cycling are flavoproteins [28]. …”

Section: Resultssupporting

confidence: 72%

Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase

Fussell

Udasin

Gray

et al. 2011

Free Radical Biology and Medicine

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“…13 CPR and its ∆1-74 catalytic domain (trCPR) were purified according to the previously reported procedures. 14,15 CYP3A4 Crystallization -∆3-22 CYP3A4 was co-crystallized with PRG at room temperature by a hanging drop vapor diffusion method. CYP3A4 (65 mg/ml) in 100 mM phosphate, pH 7.4, 20% glycerol and 100 mM NaCl was incubated with a 4-fold excess of PRG solution #11 from the Hampton Research PegIon2 kit containing 12% PEG 3350 and 4%…”

Section: Methodsmentioning

confidence: 99%

Anion-Dependent Stimulation of CYP3A4 Monooxygenase

Sevrioukova

Poulos

2015

Biochemistry

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We co-crystallized human cytochrome P450 3A4 (CYP3A4) with progesterone (PRG) under two different conditions, but the resulting complexes contained only one PRG molecule bound to the previously identified peripheral site. A novel feature in one of our structures is a citrate ion, originating from the crystallization solution. The citrate-binding site is located in an area where the N-terminus splits from the protein core and, thus, is suitable for the interaction with the anionic phospholipids of the microsomal membrane. We investigated how citrate affects the function of a soluble CYP3A4 monooxygenase system consisting of equimolar amounts of CYP3A4 and cytochrome P450 reductase (CPR). Citrate was found to affect the properties of both redox partners and stimulated their catalytic activities in a concentration-dependent manner via a complex mechanism. CYP3A4-substrate binding, reduction of CPR with NADPH, and interflavin and interprotein electron transfer were identified as citrate-sensitive steps. Comparative analysis of various negatively charged organic compounds indicated that, in addition to alterations caused by changes in ionic strength, anions modulate the properties of CYP3A4 and CPR through specific anion-protein interactions. Our results help to better understand previous observations and provide new mechanistic insights into CYP3A4 function.

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“…This investigation has provided evidence for CYP1B1 activity in RCCs and the presence of other metabolically active P450 enzymes in RCCs. Cytochrome P450 reductase is an essential component of the cytochrome P450 monoxygenase system, which catalyses the transfer of electrons from NADPH to P450 enzymes (Djordjevic et al, 1995). P450R is also involved in the bioreductive metabolism of a number of anticancer drugs including tirapazamine (Patterson et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Cytochrome P450 CYP1B1 activity in renal cell carcinoma

2004

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Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n ¼ 11). Measurable levels of active P450R were determined in all normal (n ¼ 11) and tumour samples (n ¼ 26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.

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Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

Cited by 31 publications

References 44 publications

Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase

Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase

Anion-Dependent Stimulation of CYP3A4 Monooxygenase

Cytochrome P450 CYP1B1 activity in renal cell carcinoma

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