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Lucy Waskell - One of the best experts on this subject based on the ideXlab platform.
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the fmn 140s loop of Cytochrome P450 reductase controls electron transfer to Cytochrome P450
International Journal of Molecular Sciences, 2021Co-Authors: Freeborn Rwere, Lucy WaskellAbstract:Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal Cytochrome P450s (cyt P450s). The length and sequence of the "140s" FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the "140s loop" by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-Cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the "140s loop".
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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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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.
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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, 2010Co-Authors: Lucy WaskellAbstract: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 ( 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.
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structure and function of an nadph Cytochrome P450 oxidoreductase in an open conformation capable of reducing Cytochrome P450
Journal of Biological Chemistry, 2009Co-Authors: Djemel Hamdane, Sang Choul Im, Haoming Zhang, Lucy WaskellAbstract:NADPH-Cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal Cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to Cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, Cytochrome P450. A model of the complex between an open conformation of CYPOR and Cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.
Alexander I. Archakov - One of the best experts on this subject based on the ideXlab platform.
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from electrochemistry to enzyme kinetics of Cytochrome P450
Biosensors and Bioelectronics, 2018Co-Authors: Victoria V. Shumyantseva, Tatiana V. Bulko, A V Kuzikov, Rami A Masamrekh, Alexander I. ArchakovAbstract:This review is an attempt to describe advancements in the electrochemistry of Cytochrome P450 enzymes (EC 1.14.14.1) and to study molecular aspects and catalytic behavior of enzymatic electrocatalysis. Electroanalysis of Cytochrome P450 demonstrates how to translate theoretical laws and equations of classical electrochemistry for the calculation of the kinetic parameters of enzymatic reactions and then translation of kinetic parameters to interpretation of drug-drug interactions. The functional significance of Cytochrome P450s (CYPs) includes the metabolism of drugs, foreign chemicals, and endogenic compounds. The pharmaceutical industry needs sensitive and cost-effective systems for screening new drugs and investigation of drug-drug interactions. The development of different types of CYP-based biosensors is now in great demand. This review also highlights the characteristics of electrode processes and electrode properties for optimization of the Cytochrome P450 electroanalysis. Electrochemical Cytochrome P450-biosensors are the most studied. In this review, we analyzed electrode/Cytochrome P450 systems in terms of the mechanisms underlying P450-catalyzed reactions. Screening of potential substrates or inhibitors of Cytochromes P450 by means of electrodes were described.
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photochemical properties of a riboflavins Cytochrome P450 2b4 complex
Biosensors and Bioelectronics, 2002Co-Authors: Victoria V. Shumyantseva, Tatiana V. Bulko, Rolf D. Schmid, Alexander I. ArchakovAbstract:The present study demonstrates the possible use of a non-covalent complex of riboflavins with Cytochrome P450 2B4 (artificial flavoCytochrome P450 2B4) for photo-induced intermolecular electron transfer between the isoalloxazine cycle of flavins and the ferric heme group of Cytochrome P450 2B4. Riboflavin was used as a light-induced electron donor for the transfer of electrons to Cytochrome P450. The quantitative measurement of the photocurrent, generated by photoreduction of non-covalent flavoCytochrome P450 2B4, was carried out. In the presence of typical substrates for Cytochrome P450 2B4 the decrease of cathodic photocurrent occurred, generated not only by riboflavin itself but also by a riboflavin/Cytochrome P450 complex. It was demonstrated that flavoCytochromes might serve as molecular amplifiers of a photocurrent, generated upon flavins' reduction. Introduction of flavin residues into the Cytochrome P450 molecule transformed this haemoprotein into a photoreceptor and a photodiode and, in addition, into a photosensitive and photo-activated enzyme.
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afm study of membrane proteins Cytochrome P450 2b4 and nadph Cytochrome P450 reductase and their complex formation
Archives of Biochemistry and Biophysics, 1999Co-Authors: O.i. Kiselyova, Igor V. Yaminsky, Irina P. Kanaeva, Vadim Yu. Kuznetsov, Alexander I. ArchakovAbstract:The application of the AFM technique for visualization of membrane proteins and for measuring their dimensions was demonstrated. The AFM images of the microsomal monooxygenase system components-Cytochrome P450 2B4 and NADPH-Cytochrome P450 reductase-were obtained by using two types of supports-hydrophobic, highly oriented pyrolytic graphite (HOPG) and hydrophilic mica. It was shown that hemo- and flavoprotein monomers and oligomers can be adsorbed to and visualized on HOPG. On the negatively charged mica matrix, flavoprotein oligomers dissociated to monomers while hemoprotein oligomers dissociated into less aggregated particles. The images of Cytochrome P450 2B4 and NADPH-Cytochrome P450 reductase monomers were about 3 and 5 nm high, respectively, while the images of oligomeric forms of these proteins were about 10 and 8 nm high, respectively. We were able to observe the binary complexes composed of monomeric proteins, Cytochrome P450 2B4 and its reductase and to measure the heights of these complexes (7 nm). The method is applicable for visualization of not only individual proteins but also their complexes.
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comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver i properties of nadph Cytochrome P450 reductase and Cytochrome P450 lm2 2b4 monomers
Archives of Biochemistry and Biophysics, 1992Co-Authors: Irina P. Kanaeva, Ilya R Dedinskii, Elena D Skotselyas, Arkadii G Krainev, Irina V Guleva, Irina F Sevryukova, Yakov M Koen, G P Kuznetsova, G I Bachmanova, Alexander I. ArchakovAbstract:Abstract Oligomers and monomers of NADPH-Cytochrome P450 reductase and Cytochrome P450 LM 2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-Cytochrome P450 reductase and Cytochrome P450 LM 2 oligomers were 14.8 ± 1.7 and 19.2 ± 1.4 nm, respectively. Twentyfour-hour incubation with Emulgen 913 at 4 °C at a molar ratio of 1:100 led to the monomerization of NADPH-Cytochrome P450 reductase and Cytochrome P450 LM 2 oligomers, the particle sizes diminishing to 6.1 ± 1.3 and 5.2 ± 0.4 nm, respectively. The thermal stability of NADPH-Cytochrome P450 reductase monomers was the same as that of oligomers, whereas Cytochrome P450 LM 2 monomers were less thermostable than oligomers and Cytochrome P450 in microsomes. Similar to Cytochrome P450 LM 2 oligomers and the microsomal hemoprotein, Cytochrome P450 LM 2 monomers formed complexes with type I and II substrates, but with K d values higher than those of microsomes and Cytochrome P450 LM 2 oligomers. Kinetic parameters ( V max and K m ) of H 2 O 2 - and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of Cytochrome P450 LM 2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in Cytochrome P450 LM 2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.
Haoming Zhang - One of the best experts on this subject based on the ideXlab platform.
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uncovering the role of hydrophobic residues in Cytochrome P450 Cytochrome P450 reductase interactions
Biochemistry, 2011Co-Authors: Cesar Kenaan, Haoming Zhang, Erin V Shea, Paul F HollenbergAbstract:Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, Cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.
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structure and function of an nadph Cytochrome P450 oxidoreductase in an open conformation capable of reducing Cytochrome P450
Journal of Biological Chemistry, 2009Co-Authors: Djemel Hamdane, Sang Choul Im, Haoming Zhang, Lucy WaskellAbstract:NADPH-Cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal Cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to Cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, Cytochrome P450. A model of the complex between an open conformation of CYPOR and Cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.
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Role of Cytochrome b5 in catalysis by Cytochrome P450 2B4.
Biochemical and biophysical research communications, 2005Co-Authors: Haoming Zhang, Eugene MyshkinAbstract:Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 Cytochromes. Its action is known to depend on the isozyme of Cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of Cytochrome b5 in Cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by Cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by Cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of Cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both Cytochrome b5 and Cytochrome P450 reductase interact with Cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although Cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.
Emily E Scott - One of the best experts on this subject based on the ideXlab platform.
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structure of an ancestral mammalian family 1b1 Cytochrome P450 with increased thermostability
Journal of Biological Chemistry, 2020Co-Authors: Aaron G Bart, Kurt L Harris, Elizabeth M J Gillam, Emily E ScottAbstract:: Mammalian Cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30-40 °C. Reconstructed ancestral Cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the Cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral Cytochrome P450, revealing key features that appear to contribute to its thermostability.
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comparison of antifungal azole interactions with adult Cytochrome P450 3a4 versus neonatal Cytochrome P450 3a7
Drug Metabolism and Disposition, 2018Co-Authors: Malika P Godamudunage, Anne M Grech, Emily E ScottAbstract:Adult drug metabolism is dominated by Cytochrome P450 3A4 (CYP3A4), which is often inhibited by antifungal azole drugs, resulting in potential alterations in drug metabolism and adverse drug/drug interactions. In the fetal and neonatal stages of life, the 87% identical Cytochrome P450 3A7 (CYP3A7) is expressed but not CYP3A4. Azole antifungals developed for adults are also used in neonates, assuming they interact similarly with both enzymes, but systematic information is lacking. Herein a method was developed for generating recombinant purified CYP3A7. Thirteen different azoles were then evaluated for binding and inhibition of purified human CYP3A4 versus CYP3A7. All imidazole-containing azoles bound both enzymes via coordination to the heme iron and inhibited both with IC50 values ranging from 180 nM for clotrimazole to the millimolar range for imidazole itself. Across this wide range of potencies, CYP3A4 was consistently inhibited more strongly than CYP3A7, with clotrimazole being the least selective (1.5-fold) inhibitor and econazole the most selective (12-fold). Observations for 1,2,4-triazole-containing azoles were more varied. Most bound to CYP3A4 via coordination to the heme iron, but several also demonstrated evidence of a distinct binding mode at low concentrations. However, only posaconazole inhibited CYP3A4. Of the triazoles, only posaconazole inhibited CYP3A7, again less potently than CYP3A4. Spectral evidence for binding was weak or nonexistent for all triazoles. Overall, although the details of binding interactions do vary, the same azole compounds inhibit both enzymes, albeit with weaker interactions with CYP3A7 compared with CYP3A4.
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structure based design of inhibitors with improved selectivity for steroidogenic Cytochrome P450 17a1 over Cytochrome P450 21a2
Journal of Medicinal Chemistry, 2018Co-Authors: Charlie Fehl, Emily E Scott, Caleb D Vogt, Rahul Yadav, Jeffrey AubeAbstract:Inhibition of androgen biosynthesis is clinically effective for treating androgen-responsive prostate cancer. Abiraterone is a clinical first-in-class inhibitor of Cytochrome P450 17A1 (CYP17A1) required for androgen biosynthesis. However, abiraterone also causes hypertension, hypokalemia, and edema, likely due in part to off-target inhibition of another steroidogenic Cytochrome P450, CYP21A2. Abiraterone analogs were designed based on structural evidence that B-ring substituents may favorably interact with polar residues in binding CYP17A1 and sterically clash with residues in the CYP21A2 active site. The best analogs increased selectivity of CYP17A1 inhibition up to 84-fold compared with 6.6-fold for abiraterone. Cocrystallization with CYP17A1 validated the intended new contacts with CYP17A1 active site residues. Docking these analogs into CYP21A2 identified steric clashes that likely underlie decreased binding and CYP21A2 inhibition. Overall, these analogs may offer a clinical advantage in the form of reduced side effects.
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structures of Cytochrome P450 3a4
Trends in Biochemical Sciences, 2005Co-Authors: Emily E Scott, James R HalpertAbstract:Cytochrome P450 3A4 (CYP3A4) catalyzes the initial step in the clearance of many pharmaceuticals and foreign chemicals. The structurally diverse nature of CYP3A4 substrates complicates rational prediction of their metabolism and identification of potential drug interactions. The first molecular structures of human CYP3A4 were recently determined, revealing an active site of sufficient size and topography to accommodate either large ligands or multiple smaller ligands, as suggested by the heterotropic and homotropic cooperativity of the enzyme.
C R Wolf - One of the best experts on this subject based on the ideXlab platform.
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dissection of nadph Cytochrome P450 oxidoreductase into distinct functional domains
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: G Smith, C R WolfAbstract:NADPH-Cytochrome P450 oxidoreductase transfers electrons from NADPH to Cytochrome P450 and catalyzes the one-electron reduction of many drugs and foreign compounds. This enzyme is a flavoprotein containing the cofactors FMN and FAD, which are essential for its function. We have expressed the putative FMN and FAD/NADPH binding domains of P450 reductase and show that these distinct peptides fold correctly to bind their respective cofactors. The FAD/NADPH domain catalyzed the one-electron reduction of a variety of substrates but did not efficiently reduce Cytochrome c or Cytochrome P450 (as judged by the oxidation of the CYP1A1 substrate 7-ethoxyresorufin). However, the domains could be combined to provide a functional enzyme active in the reduction of Cytochrome c and in transferring electrons to Cytochrome P450. Both the reconstitution of the domains and the direct binding of Cytochrome c to the FMN domain were ionic-strength dependent. The FMN domain containing the hydrophobic membrane anchor sequence was a potent inhibitor of reconstituted monooxygenase activity. These data strongly support the hypothesis that FMN/FAD-containing proteins have evolved as a fusion of two ancestral genes and provide fundamental insights into how this and structurally related proteins, such as nitric oxide synthase and sulfite reductase, have evolved and function.
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the role of Cytochrome P450 and Cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di n oxide hypoxic cytotoxin 3 amino l 2 4 benzotriazine l 4 dioxide sr 4233 win 59075 by mouse liver
Biochemical Pharmacology, 1992Co-Authors: Michael I Walton, C R Wolf, Paul WorkmanAbstract:Abstract SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of Cytochrome P450 and Cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono- N -oxide SR 4317 (3-amino-1,2, 4-benzotriazine-l-oxide) is NADPH. enzyme and hypoxia dependent. An inhibitory antibody to Cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat Cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20–30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific Cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of Cytochrome P450 involvement was provided by the use of other P450 ligands: β- diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n - octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-a-carbonitrile and β-napthoflavone, suggesting that Cytochrome P450 subfamilies IIB. IIC and IIIA may be involved. Since Cytochrome P450 and P450 reductase catalyse roughly 70 and 30%. of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug.
