The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Lucy Waskell - One of the best experts on this subject based on the ideXlab platform.
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lipid exchange in nanodiscs discloses membrane boundaries of Cytochrome P450 Reductase
Chemical Communications, 2018Co-Authors: Carlo Barnaba, Lucy Waskell, Thirupathi Ravula, Ilce G Medinameza, G M Anantharamaiah, Ayyalusamy RamamoorthyAbstract:Lipids are critical for the function of membrane proteins. NADPH-Cytochrome-P450-Reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil Cytochrome-P450-Reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.
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protonation of the hydroperoxo intermediate of Cytochrome P450 2b4 is slower in the presence of Cytochrome P450 Reductase than in the presence of Cytochrome b5
Biochemistry, 2016Co-Authors: Naw May Pearl, Sang Choul Im, Jarett Wilcoxen, Ryan C Kunz, Joseph E Darty, David R Britt, Stephen W Ragsdale, Lucy WaskellAbstract:Microsomal Cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by Cytochrome P450 Reductase, the second electron can also be donated by Cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5. To improve our understanding of the molecular basis of the stimulatory effect of b5 and to test the hypothesis that b5 stimulates catalysis by more rapid protonation of the anionic ferric hydroperoxo heme intermediate of P450 (Fe3+OOH)− and subsequent formation of the active oxidizing species (Fe+4═O POR•+), we have freeze-quenched the reaction mixture during a single turnover following reduction of oxyferrous P450 2B4 by each of its redox partners, b5 and P450 Reductase. The electron paramagnetic resonance spectra of the freeze-quenched reaction mixtures lacked evidence of a hydroperoxo intermediate when b5 was the reductant presumably because hydroperoxo protonation and catalysis occurred within...
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kinetic and structural characterization of the interaction between the fmn binding domain of Cytochrome P450 Reductase and Cytochrome c
Journal of Biological Chemistry, 2015Co-Authors: Rui Huang, Lucy Waskell, Meng Zhang, Freeborn Rwere, Ayyalusamy RamamoorthyAbstract:Cytochrome P450 Reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis.
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Distinct conformational behaviors of four mammalian dual-flavin Reductases (Cytochrome P450 Reductase, methionine synthase Reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles
FEBS Journal, 2014Co-Authors: Mohammad Mahfuzul Haque, Mekki Bayachou, Jesús Tejero, Claire Kenney, Naw May Pearl, Sang Choul Im, Lucy Waskell, Dennis J. StuehrAbstract:: Multidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoReductases that catalyze important biological electron transfer reactions: Cytochrome P450 Reductase, methionine synthase Reductase and endothelial and neuronal nitric oxide synthase. We used stopped-flow spectroscopy, single turnover methods and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. The results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12% to 72% open state) and also vary 100-fold with respect to their rates of conformational switching between unreactive closed and reactive open states (Cytochrome P450 Reductase > neuronal nitric oxide synthase > methionine synthase Reductase > endothelial nitric oxide synthase). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (Cytochrome c Reductase activity) based on its unique conformational setpoint and switching rates. The present study is the first to quantify these conformational parameters among the diflavin enzymes and suggests how the parameters might be manipulated to speed or slow biological electron flux.
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the interaction of microsomal Cytochrome P450 2b4 with its redox partners Cytochrome P450 Reductase and Cytochrome b5
Archives of Biochemistry and Biophysics, 2011Co-Authors: Sang Choul Im, Lucy WaskellAbstract:Abstract Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other Cytochromes P450. The Cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by Cytochrome P450 Reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either Cytochrome P450 Reductase or Cytochrome b 5 . In both instances, product formation occurs. When the second electron is donated by Cytochrome b 5 , catalysis (product formation) is ∼10- to 100-fold faster than in the presence of Cytochrome P450 Reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b 5 has also been shown to compete with Cytochrome P450 Reductase for a binding site on the proximal surface of Cytochrome P450 2B4. These two different effects of Cytochrome b 5 on Cytochrome P450 2B4 reactivity can explain how Cytochrome b 5 is able to stimulate, inhibit, or have no effect on Cytochrome P450 2B4 activity. At low molar ratios ( b 5 to Cytochrome P450 Reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of Cytochrome b 5 to Cytochrome P450 Reductase, Cytochrome b 5 inhibits activity by binding to the proximal surface of Cytochrome P450 and preventing the Reductase from reducing ferric Cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of Cytochrome b 5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that Cytochrome b 5 stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of Cytochrome P450 to be formed more rapidly than in the presence of Reductase.
Stephen W Ragsdale - One of the best experts on this subject based on the ideXlab platform.
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protonation of the hydroperoxo intermediate of Cytochrome P450 2b4 is slower in the presence of Cytochrome P450 Reductase than in the presence of Cytochrome b5
Biochemistry, 2016Co-Authors: Naw May Pearl, Sang Choul Im, Jarett Wilcoxen, Ryan C Kunz, Joseph E Darty, David R Britt, Stephen W Ragsdale, Lucy WaskellAbstract:Microsomal Cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by Cytochrome P450 Reductase, the second electron can also be donated by Cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5. To improve our understanding of the molecular basis of the stimulatory effect of b5 and to test the hypothesis that b5 stimulates catalysis by more rapid protonation of the anionic ferric hydroperoxo heme intermediate of P450 (Fe3+OOH)− and subsequent formation of the active oxidizing species (Fe+4═O POR•+), we have freeze-quenched the reaction mixture during a single turnover following reduction of oxyferrous P450 2B4 by each of its redox partners, b5 and P450 Reductase. The electron paramagnetic resonance spectra of the freeze-quenched reaction mixtures lacked evidence of a hydroperoxo intermediate when b5 was the reductant presumably because hydroperoxo protonation and catalysis occurred within...
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protein protein interactions in the mammalian heme degradation pathway heme oxygenase 2 Cytochrome P450 Reductase and biliverdin Reductase
Journal of Biological Chemistry, 2014Co-Authors: Andrea Morris Spencer, Ireena Bagai, Donald F Becker, Erik R P Zuiderweg, Stephen W RagsdaleAbstract:Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via Cytochrome P450 Reductase (CPR). Biliverdin Reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence.
Lee-young Chau - One of the best experts on this subject based on the ideXlab platform.
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Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/Cytochrome P450 Reductase interaction with anti-tumor activity
Journal of Biomedical Science, 2018Co-Authors: Yi-tai Chou, Fu-fei Hsu, Ying-chih Chen, Yuan-hao Hsu, John T.-a. Hsu, Lee-young ChauAbstract:Background Heme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and Cytochrome P450 Reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy. Methods We employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay. Results Danthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells. Conclusions These findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.
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identification of danthron as an isoform specific inhibitor of heme oxygenase 1 Cytochrome P450 Reductase interaction with anti tumor activity
Journal of Biomedical Science, 2018Co-Authors: Yi-tai Chou, Fu-fei Hsu, Ying-chih Chen, Yuan-hao Hsu, John T.-a. Hsu, Lee-young ChauAbstract:Heme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and Cytochrome P450 Reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy. We employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay. Danthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells. These findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.
Roland C Wolf - One of the best experts on this subject based on the ideXlab platform.
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role of hepatic Cytochromes P450 in bioactivation of the anticancer drug ellipticine studies with the hepatic nadph Cytochrome P450 Reductase null mouse
Toxicology and Applied Pharmacology, 2008Co-Authors: Marie Stiborová, Michaela Moserová, David H Phillips, Volker M Arlt, Colin J Henderson, Roland C Wolf, Věra Kotrbova, Jiři Hudecek, Eva FreiAbstract:Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by Cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which Cytochrome P450 oxidoReductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by P-32-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study. (c) 2007 Elsevier Inc. All rights reserved.
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relationship between hepatic phenotype and changes in gene expression in Cytochrome P450 Reductase por null mice
Biochemical Journal, 2005Co-Authors: Xiu Jun Wang, Colin J Henderson, Mark Chamberlain, Olga Vassieva, Roland C WolfAbstract:Cytochrome P450 Reductase is the unique electron donor for microsomal Cytochrome P450s; these enzymes play a major role in the metabolism of endogenous and xenobiotic compounds. In mice with a liver-specific deletion of Cytochrome P450 Reductase, hepatic Cytochrome P450 activity is ablated, with consequent changes in bile acid and lipid homoeostasis. In order to gain insights into the metabolic changes resulting from this phenotype, we have analysed changes in hepatic mRNA expression using microarray analysis and real-time PCR. In parallel with the perturbations in bile acid levels, changes in the expression of key enzymes involved in cholesterol and lipid homoeostasis were observed in hepatic Cytochrome P450 Reductase null mice. This was characterized by a reduced expression of Cyp7b1, and elevation of Cyp7a1 and Cyp8b1 expression. The levels of mRNAs for other Cytochrome P450 genes, including Cyp2b10, Cyp2c29, Cyp3a11 and Cyp3a16, were increased, demonstrating that endogenous factors play a role in regulating the expression of these proteins and that the increases are due, at least in part, to altered levels of transcripts. In addition, levels of mRNAs encoding genes involved in glycolysis and lipid transport were also increased; the latter may provide an explanation for the increased hepatic lipid content observed in the hepatic null mice. Serum testosterone and oestradiol levels were lowered, accompanied by significantly decreased expression of Hsd3b2 (3β-hydroxy-Δ5-steroid dehydrogenase-2), Hsd3b5 (3β-hydroxy-Δ5-steroid dehydrogenase-5) and Hsd11b1 (11β-hydroxysteroid dehydrogenase type 1), key enzymes in steroid hormone metabolism. These microarray data provide important insights into the control of metabolic pathways by the Cytochrome system.
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inactivation of the hepatic Cytochrome P450 system by conditional deletion of hepatic Cytochrome P450 Reductase
Journal of Biological Chemistry, 2003Co-Authors: Colin J Henderson, Diana M E Otto, Dianne Carrie, Mark A Magnuson, Aileen W Mclaren, Ian Rosewell, Roland C WolfAbstract:Abstract Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein Reductase (EC 1.6.2.4, Cytochrome P450 Reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivometabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.
Birger Lindberg Moller - One of the best experts on this subject based on the ideXlab platform.
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monitoring shifts in the conformation equilibrium of the membrane protein Cytochrome P450 Reductase por in nanodiscs
Journal of Biological Chemistry, 2012Co-Authors: Maria Wadsater, Tomas Laursen, Birger Lindberg Moller, Aparajita Singha, Nikos S Hatzakis, Dimitrios Stamou, Robert Barker, Kell Mortensen, R Feidenhansl, Marite CardenasAbstract:Abstract Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored Cytochrome P450 Reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to Cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 A, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.
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conformational changes of the nadph dependent Cytochrome P450 Reductase in the course of electron transfer to Cytochromes P450
Biochimica et Biophysica Acta, 2011Co-Authors: Tomas Laursen, Kenneth Jensen, Birger Lindberg MollerAbstract:Abstract The NADPH-dependent Cytochrome P450 Reductase (CPR) is a key electron donor to eucaryotic Cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR undergoes large conformational changes. This mini-review discusses the new evidence provided for such conformational changes involving a combination of a “swinging” and “rotating” model and highlights the molecular mechanisms by which formation of these conformations are controlled and thereby enables CPR to serve as an effective electron transferring “nano-machine”.
