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1
Skinner, Michael Stephen. "Olfactory cytochrome P450". Thesis, University of Warwick, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307349.
Testo completo
2
Farooq, Yassar. "Mechanisms of electron transfer from cytochrome P450 reductase to cytochrome P450 3A4". Thesis, University of Leicester, 2010. http://hdl.handle.net/2381/8597.
Testo completoAbstract (sommario):
The study demonstrates that CPR and P450 3A4 can be prepared to highly pure state by the use of detergent CHAPS. Optimisation of published methods led to pure flavoprotein, ~90% full-length with a small amount of truncated CPR. The reconstitution of CPR and P450 3A4 into liposomes using CHAPS and Superose 6 column purification has achieved a homogenous highly functional proteoliposomes with good catalytic activity and almost completely reducible P450 3A4 in a simple controllable system. The spectroscopic data has shown that reduction of P450 3A4 in proteoliposomes was at least 10 fold higher than in the simple reconstituted system suggesting that isolation of proteoliposomes from unbound protein aggregates had marked effect on the catalytic activity of P450. Negative staining electron microscopy has revealed proteoliposomes of having mean diameter of 200 ±15 nm in size; the lipid:protein ratio indicated that they incorporated 350 proteins per liposome. Type 1 difference spectral changes were observed upon binding of testosterone and erythromycin. Measurements of the first electron transfer have shown that the reduction of P450 3A4 is highly dependent on the presence of substrate. P450 3A4 reduction in proteoliposomes in the presence of testosterone was rapid and biphasic, with 90% of the P450 reduced in the fast phase, whereas reduction in the presence of erythromycin was monophasic, but substantially slower. Changes in the CPR:P450 molar ratio did not alter the rate of reduction and thus the data strongly indicates that first electron transfer occurs through preformed CPR:P450 complexes in the proteoliposomes that have a lifetime of about order of hundreds of milliseconds. The origin of the slow phase of reduction of P450 is not conclusive from our experiments, but it may be due to P450 heterogeneity. NADPH oxidation and 6β-hydroxytestosterone formation results have revealed that P450 3A4 is highly uncoupled enzyme with rates limited by CPR to P450 3A4 ratio. Hyperbolic plots of rates of NADPH consumption and 6β hydroxytestosterone formation vs CPR concentration indicate apparent Ks of 0.4 μM. This suggests that CPR:P450 complex can dissociate and reform between first and second electron transfer, which in turn indicates that second electron transfer occurs by diffusion mechanism.
3
Al-Anizy, Mohammed. "Studies on cytochrome P450 4X1". Thesis, University of Nottingham, 2005. http://eprints.nottingham.ac.uk/10404/.
Testo completoAbstract (sommario):
Antisera raised against recombinant human CYP4Z1 (4Z1), mouse Aryl hydrocarbon receptor (AhR), and C.elegans Latrophilin (LpH) proteins were titred over the course of several bleeds. The sensitivity of the antibodies shows an increase over the course of several immunizations, with the minimum amount detected being 1 ng of 4Z1, 0.3 ng of AhR, and 0.3 ng of LpH. Terminal bleeds were taken for the AhR and LpH antisera. The AhR antisera detects proteins in rat and mouse liver cytosol consistent with previous reports of the AhR. LpH is predicted to be localized in a membrane compartment on the basis of its primary structure, so sub-cellular fractions of C.elegans were isolated and tested, revealing a protein of ~113 kDa in a crude membrane preparation. This protein was not solubilized in < 1% Emulgen 913, but was soluble in 2% dodecylmaltoside. A fresh 15k supernatant treated with 2% dodecylmaltoside showed a strong band of LpH protein at ~113 kDa. However, 66 kDa band was detected when the samples were stored overnight at 4 degrees centigrade due to presence of a G protein coupled receptor proteolysis site (GPS) between position M536 and C547 of LpH, which is a characteristic feature in the LpH protein family. In order to study the expression of the CYP 4X1 in mouse tissues, RNase protection assays were performed. Different tissues were assayed at the same time and the same riboprobe was used to hybridise all the samples. The CYP 4\x1 probe was shown to be full length (-ve control). However, the +ve control (RNase positive) shows an absence of signal when hybridised to the yeast tRNA demonstrating the specificity of the signal in the samples. Several RNA samples were hybridised with mouse cyp4X1 gene probe, such as aorta, brain and heart and liver. The mouse cyp4X1 gene appears to have 12 exon from the genomic sequence and encodes a protein which high identity with the human and rat cyp4X1 gene. The full-length of the probe was 424 b.p and the protected fragments were 177 b.p. The murine cyp4X1 was not expressed in control liver, but is expressed in brain at high level. Cyp4X1 gene was also investigated in aorta tissue and found to be expressed at low levels. Known inducers of hepatic cytochrome P450 were used (Ciprofibrate, TCDD, PB, and dexamethasone), but had no induction effect in the samples. Western blotting of brain confirmed that the cyp4X1 protein is expressed in brain, and quantification showed that this is a major cytochrome P450 in brain.
4
Bell, Stephen Graham. "The use of active site mutants of cytochrome P450(cam) in chemical synthesis". Thesis, University of Oxford, 1999. http://ora.ox.ac.uk/objects/uuid:7f48cf79-37b0-45cd-a40e-e971af466cff.
Testo completoAbstract (sommario):
This thesis describes a study of the substrate selectivity of active site mutants of the monooxygenase cytochrome P450cam. A range of mutants was constructed which replaced the phenolic side-chain at the Tyr-96 position by various hydrophobic amino acid residues. These 'hydrophobic mutants' were then combined with other mutations around the active site (Val-247, Phe-87, Ile-395 and Phe-193) which altered the space available at different positions in the active site. These mutants were then tested with an in vitro reconstituted P450cam system with a range of substrates related to diphenylmethane and phenylcylcohexane. All of these large compounds were poor substrates for the wild-type enzyme. It was found that it was necessary to increase both the space available in the active site and the active site hydrophobicity to achieve substrate turnover. The substrates were oxidised preferentially on the aliphatic cyclohexyl ring over the more constrained phenyl ring suggesting that the active site is predisposed to binding the cyclohexyl ring close to the haem. Hydroxylation using the in vitro reconstituted P450cam system is limited by catalyst lifetime and the need for the expensive cofactor NADH. For P450cam hydroxylation to become a viable synthetic method it is necessary to find ways to bypass the use of NADH. For this reason various self-sufficient P450cam system were constructed and expressed in E. coli. The best of these, despite limited protein expression, was found to turnover camphor with the wild-type P450cam enzyme and other substrates with the Y96A mutant. The in vivo catalytic system was then used to screen many P450cam mutants for the oxidation of natural products, monoterpenes and sesquiterpenes (e.g. limonene, pinene and valencene). Most of the target substrates are not oxidised by the wild-type enzyme but all are hydroxylated by some if not all of the P450cam mutants with different degrees of selectivity. Some of the products identified so far are important compounds in the field of flavour and fragrance chemistry (e.g. verbenol and nookatone).
5
Sideri, Anastasia. "Directed evolution of cytochrome P450 BM3". Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435931.
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6
Marsh, Rachael. "Cytochrome P450 studies in Aspergillus fumigatus". Thesis, University of Sheffield, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364267.
Testo completo
7
Maughan, Juanita Amanda. "Molecular investigations of plant cytochrome P450". Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388204.
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8
Papagiannidou, Eleni. "Cytochrome P450-mediated metabolism of melatonin". Thesis, University of Surrey, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422928.
Testo completo
9
Tyzack, Jonathan David. "Prediction of cytochrome P450 xenobiotic metabolism". Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708289.
Testo completo
10
Busi, Florent. "Pharmacogénétique du cytochrome P450 humain CYP3A5". Paris 5, 2005. http://www.theses.fr/2005PA05P621.
Testo completoAbstract (sommario):
Les CYP3A sont les P450 les plus abondants dans le foie où ils métabolisent plus de 50 % des médicaments. Le CYP3A5 n'est exprimé que dans 25 % de la population caucasienne. Une mutation (CYP3A5*3) entraînant un épissage alternatif a été associée à un faible niveau de CYP3A5. Un niveau d'ARNm CYP3A5 plus élevé chez les individus *1/*3 par rapport aux homozygotes *3/*3 nous a amené à rechercher les causes de cette variation. L'ARN *3 est plus instable et sa dégradation est réalisée par la NMD (nonsense mediated mRNA decay). Le génotype CYP3A5 a des conséquences en clinique. Nous avons mis au point une technique de génotypage informationnel basé sur la PCR quantitative permettant de déterminer en une seule réaction le génotype et le phénotype CYP3A5. Cette technique permettrait d'ajuster les doses de molécules de faible index thérapeutique lorsque leur pharmacocinétique dépend du CYP3A5. Cette technique peut être généralisée à l'étude des gènes épissés alternativementCYP3A are the most abundant P450 in the human liver where they metabolize more than 50% of drugs. CYP3A5 is found in only 25% of the Caucasian population. A SNP (CYP3A5*3) yielding to alternative splicing was associated with low CYP3A5 protein content. A higher CYP3A5 mRNA level found in *1/*3 as compared to *3/*3 individuals led us to investigate the mechanism underlying this difference. CYP3A5*3 mRNA was unstable and its degradation was mediated by nonsense mediated mRNA decay (NMD). CYP3A5 genotype has consequences in clinics. We describe a new informative genotyping assay based on quantitative real-time PCR, allowing the determination of CYP3A5 genotype and phenotype in one single step. This assay could be used to improve the dosage of drugs metabolized by CYP3A5 having a narrow therapeutic window. This assay can be generalized to the study of alternatively spliced genes
11
Eiben, Sabine. "CYP102A P450 monooxygenases: comparative analysis and construction of cytochrome P450 chimera". [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-33683.
Testo completo
12
Bertrand-Thiébault, Céline. "Cytochromes P450 et tonus vasculaire". Nancy 1, 2004. https://tel.archives-ouvertes.fr/tel-00120305.
Testo completoAbstract (sommario):
Les cytochromes P450 (CYP) sont des enzymes dont le rôle principalement connu est la détoxification. Cependant leur activité peut conduire à la formation de métabolites ayant des effets pharmacologiques, toxicologiques ou physiologiques. Depuis quelques années le rôle des cytochromes P450 dans le métabolisme de l'acide arachidonique est bien décrit. Parmi les métabolites produits lors de cette biotranformation, les acides epoxyeicosatriènoi͏̈ques (les EETs) et les acides hydroxyeicosatetraènoi͏̈ques (les HETEs ) occupent une place importante dans la régulation du tonus vasculaire. Ainsi les EETs ont des propriétés vasodilatatrices tandis que les HETEs ont des propriétés surtout vasoconstrictrices. Au cours de ce travail, nous avons étudié l'expression de certains cytochromes P450 dans les veines saphènes humaines et nous avons caractérisé le profil d'expression de ces enzymes dans différents types cellulaires contenus dans les parois vasculaires. Nous avons montré que l'expression de la plupart des cytochromes P450 notamment CYPlBl, CYP2C, CYP2El, CYP2J2 et CYP3A était augmentée dans les échantillons de veines pathologiques humaines. Nous avons ensuite étudié dans une lignée de cellules endothéliales humaines (ECV304) et une lignée d'hépatomes d'origine humaine (HepG2), la régulation par quelques statines de certains cytochromes P450 dont les CYP2C, occupant une place importante dans la régulation de l'homéostasie vasculaire. Les statines sont des molécules utilisées dans le traitement de l'hypercholestérolémie présentant des effets plei͏̈otropiques notamment sur l'état vasculaire. Nous avons observé que l'atorvastatine était la molécule ayant le plus d'effet sur l'expression des cytochromes P450 2C8, 2C9, 2J2, 3AS et 3A7. En effet, cette molécule diminue l'expression des différents cytochromes P450 dans les cellules ECV304 alors qu'elle augmente leur expression dans les cellules HepG2. Les statines influencent également l'expression de deux types de récepteurs nucléaires impliqués dans la régulation des cytochromes P450. Ainsi, la fluvastatine augmente l'expression des récepteurs nucléaires CAR et PXR. Enfin, nous avons étudié l'effet du polymorphisme d'un cytochrome P450 de la famille 2C sur la pression sanguine et sur les marqueurs inflammatoires des pathologies cardiovasculaires. Nous avons observé que les valeurs de pressions sanguines ne variaient pas en fonction du polymorphisme de CYP2Cl9. En revanche, les taux plasmatiques d'interleukine-6 sont plus élevés chez les porteurs de l'allèle muté de CYP2Cl9*2. De la même façon, les taux plasmatiques des paramètres lipidiques tels que les triglycérides sont également plus élevés chez les mêmes sujets. Ainsi, une modification de l'expression des cytochromes P450 peut jouer un rôle dans la pathologie variqueuse et dans la pathologie vasculaire à composante inflammatoire. Cette expression peut être modulée par les statines. Enfin, le polymorphisme CYP2Cl9*2 est corrélé avec la composante inflammatoire des pathologies variqueuses probablement par l'intermédiaire des métabolites de l'acide arachidonique. Ces résultats posent alors la question de la modulation de la production des molécules vaso-actives issues de l'acide arachidonique dans les pathologies vasculaires et d'éventuels traitements par les molécules inhibitrices de l'HMGCoA réductase ou par des inhibiteurs de cytochromes P450.
13
Vergères, Guy Vergeres Guy. "The membrane topoloy of microsomal cytochrome P450 /". [S.l.] : [s.n.], 1989. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=8866.
Testo completo
14
Givens, Raymond Carlos Maeda Nobuyo. "Physiologic effects of cytochrome P450 3A activity". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1371.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Nutrition, School of Public Health." Discipline: Nutrition; Department/School: Public Health.
15
Rice, M. J. "Synthetic models of cytochrome P450 and photosynthesis". Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233252.
Testo completoAbstract (sommario):
The work presented in this dissertation describes the prepartion and characterisation of various strapped porphyrin compounds which are designed to model cytochrome P450 and the charge separation in photosynthesis. The major part of this work is concerned with models of cytochrome P450. After a brief introduction to the philosophy of modelling, there follows a review of work on the enzyme and various compounds which are designed to reproduce the enzymic characteristics. The synthesis of two singly bridged porphyrins is reported. These incorporate a pendent methyl ester in the centre of the strap and aim to mimic the proposed acylation step in the catalytic cycle. Evaluation as models was performed by a series of experiments involving the addition of potassium superoxide to the manganese complexes of these compounds. Characterisation of the mode of reactivity required the use of many physical techniques and necessitated the synthesis of a radio-labelled sample of one of the porphyrins. The results obtained suggest that superoxide binds preferentially to the bridge-free face of the macrocycle. Doubly bridged analogues of the above models were prepared which force the two faces of the porphyrin to be equivalent. Superoxide binding studies indicated a different mode of reactivity to the singly bridged models, for one of the compounds, and experiments to distinguish between possible interpretations of the results are suggested. A crown ether thiolate doubly bridged porphyrin was prepared as a model for the carbon monoxide complex of the enzyme. This was characterised by ultraviolet spectroscopy and attempts to produce a stable crystalline adduct are described. The remaining part of this work concerns a model for the charge separation process in photosynthesis. A discussion of natural systems and previous models is followed by a description of a tetraene pyromellitimide doubly bridged porphyrin, which shows significant quenching of the porphyrin fluorescence.
16
Holmes, Victoria. "Structure activity relationships of cytochrome P450 4A1". Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289361.
Testo completo
17
Sabzevari, Omid. "Azole antifungal drugs and cytochrome P450 induction". Thesis, University of Surrey, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359878.
Testo completo
18
Singh, Subir. "Role of cytochrome P450 in breast carcinogenesis". Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/role-of-cytochrome-p450-in-breast-carcinogenesis(ed2c5c1b-d2e9-458b-acc5-3c320cf22ee6).html.
Testo completoAbstract (sommario):
Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.
19
Dash, Hayley. "Studies of cytochrome P450 in biomimetic films". Thesis, University of Bath, 2007. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512273.
Testo completoAbstract (sommario):
Cytochrome P450 enzymes are a super family of haem-containing mono-oxygenases which are found in all kingdoms of life. They show extraordinary diversity in their reaction chemistry and are involved in the biotransformation of a plethora of both exogenous and endogenous compounds. There has been a variety of approaches for the immobilisation of these biological redox systems and direct electrochemistry of these proteins can go towards providing biomimetic environments for fundamental studies together with a basis for designing devices without the need for electron transfer mediators. The incorporation of these proteins into mesoporous films or onto various electrode surfaces has generated interest due to the possibility of direct electron exchange between the proteins redox active sites and the host electrode. Here, two different P450 protein systems were investigated. The water soluble P450cam or Cyp101, from the soil dwelling bacteria Pseudomonas Putida and Cyp6g1, a microsomal protein form the fruit fly Drosophila Melanogaster. In this work, firstly the Cyp101 proteins were expressed and purified from a microbial culture starting material. The various steps in the purification process freed the protein from a confining matrix followed by the separation of the protein and non-protein parts of the mixture. In the latter system the enzyme is already embedded into a microsomal unit, thus more likely to mimic a biologically active environment. The utilisation of methoxy-resorufin ether (MRES) is described as an electrochemical probe for investigating the activity of the microsomal protein. This substrate also exhibits fluorescent properties which provided a dual detection system for the enzymes activity. The work then went on to investigate the absorption and reactivity of Cyp101 in porous nanoparticulateTiO2 film electrodes and on an edge plane graphite electrode.
20
Smith, Gillian. "Xenobiotic regulation of cytochrome P450 gene expression". Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/20797.
Testo completoAbstract (sommario):
Cytochromes P450 (P450) are a ubiquitous class of monooxygenases located in the endoplasmic reticulum of mammalian cells. These enzymes catalyse the Phase I oxidative metabolism of a wide range of structurally diverse chemicals resulting in increased hydrophilicity and excretion. Certain chemicals are, however, metabolically activated by cytochrome P450, leading to the formation of cytotoxic and/or carcinogenic metabolites. This has been exploited in the design of many prodrugs, including anti-tumour agents, which are inactive as administered but become active in vivo following metabolism by one or more of the P450 isozymes. The regulation of P450 gene expression has been well documented in experimental animals, but at present there is very little information available about the regulation of human P450 genes, particularly in extra-hepatic tissues. Regulation of P450 expression by a range of xenobiotics, known to have profound effects on the expression of rodent P450 genes, has been studied in a mouse model and in cultured cells. Of particular interest were the potent and pleiotropic effects on murine P450 expression of TCPOBOP (1,4 bis [s,(3,5-dichloropyridyloxy)] benzene), which showed marked tissue and species specificity in its inductive effects in rodents. A model was developed, using human tumours grown as xenografts in immune deficient mice, in which the in vivo regulation of human P450 genes could be examined. TCPOBOP was shown to be equally effective at influencing human P450 gene expression and, in most cases, the patterns of gene regulation observed in experimental animals were also seen in the human tumours. These studies suggest that modulation of intra-tumour P450 levels by agents such as TCPOBOP may lead to enhanced metabolism of anticancer drugs such as cyclophosphamide, which require P450-mediated activaion in order to exert their anti-tumour effects.
21
Luciakova, Dominika. "Characterisation of novel cytochrome P450-fusion enzymes". Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-novel-cytochrome-p450fusion-enzymes(08d9f0eb-666c-4f0f-b3ad-1fbf52555a0e).html.
Testo completoAbstract (sommario):
This study focuses on the characterisation of three novel cytochrome P450-partner (P450-fusion) enzymes of unknown structure and function. Despite several well-established P450 functions, new structures of P450s are published frequently, with the P450-redox partner fusion systems being among the most discussed, due to their enhanced activity and biotechnological potential. Other, more intriguing, P450-fusions involve partners with functions distinct from electron transfer. Understanding why evolution drove the ‘partner’ proteins to evolve into a single unit is often unclear, but provides an important challenge for the understanding of the breadth of biochemical reactions mediated by P450s. The first P450-fusion analysed (Chapter 3) is CYP116B1 from a soil bacterium, Cupriavidus metallidurans, that displays important environmental implications. The enzyme was characterised as a functional fusion, composed of three domains: a P450 from the CYP116B family, and a phthalate dioxygenase reductase (PDOR)-like reductase binding FMN and a 2Fe-2S cluster. CYP116B1 is a stable, cytosolic enzyme but can undergo FMN cofactor loss. Studies included redox potentiometry of the intact fusion and its individual domains using spectro-electrochemical and EPR methods to enable the determination of midpoint redox potentials for individual cofactors. The CYP116B1 EPR signature was shown to be typical of P450s, and changed upon binding heme-coordinating inhibitors of the azole class. Extensive compound library screening did not reveal a substrate-like physiological “hit”. However, catalytic activity was detected towards selected thiocarbamate herbicides. GC-MS data revealed the enzymatic mechanism of herbicide degradation. The second system studied (Chapter 4) is P450-CAD, an atypical fusion of an uncharacterised soluble P450 and a cinnamyl alcohol dehydrogenase (ADH) module from Streptomyces ghanaensis; a member of the major antibiotic producing genus of bacteria. The CAD module appears unlikely to be a redox partner, but instead possibly mediates substrate/product exchange with the P450. The intact fusion was shown to aggregate during extraction. Genetic dissection of domains revealed that this was due to the highly insoluble ADH moiety. The heme domain (HD) was soluble and was characterised extensively. The enzyme displays an unusual spectrum when in the FeII-CO complex (Amax = 445 nm). The P450-CAD HD catalytic activity is supported by heterologous redox partners (E. coli flavodoxin reductase [FldR] and flavodoxin [FldA], and spinach ferredoxin reductase [FdR] and ferredoxin [Fdx]). The CAD-HD binds fatty acid substrates of carbon chain length C8-14, with the highest affinity for 12-methylmyristic acid (12M14C acid), the C12 lauric acid, its aldehyde and alcohol, indicating that the terminal methyl group is important for binding to the enzyme. Unusually, the CAD-HD also binds a range of detergent compounds. Further analysis included SEC-MALLS, thermostability and structural studies. The final enzyme studied (Chapter 5) is the P450-BDOR (a P450 linked to a benzoate dioxygenase reductase) redox-partner fusion. The unconventional trait of this enzyme is the inclusion of an FCD (a fatty acid metabolism regulator protein [FadR] C-terminal DNA-binding domain). From the point of view of P450s, DNA interaction would represent an unprecedented function, suggesting novel functions for a P450 enzyme. Thus, this enzyme requires extensive research with the expectations that new information will contribute to an expansion of knowledge of P450 diversity. This study provides initial analytical insights into the P450-BDOR system, supported with functional and kinetic data on the P450 and its reductase domain.
22
Robinson, Jacob. "Characterisation of novel cytochrome P450 fusion systems". Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-novel-cytochrome-p450-fusion-systems(5b0847b2-8a5d-427d-9434-11a50f24311c).html.
Testo completoAbstract (sommario):
The biophysical and spectroscopic characterisation of two novel P450 fusion enzymes is reported. The first of these is CYP102A3, which is a fusion of P450 haem and cytochrome P450 reductase (CPR)-like domains and functions as a catalytically self-sufficient fatty acid hydroxylase in its host organism Bacillus subtilis. The elucidation of structural aspects of the isolated haem domain of CYP102A3 (HDCYP102A3) is described. This reveals a strong homology between HDCYP102A3 and the haem domain of the related, well studied enzyme CYP102A1 (known as BM3). Examination of the substrate binding and redox properties of HDCYP102A3 reveals variations in substrate selectivity and the influence of substrate binding over the haem-iron redox potential compared to BM3. Of particular note is the apparent cooperative binding profile displayed for some branched chain fatty acid substrates with CYP102A3. The second system characterised is CYP116B1 from Cupriavidus metallidurans, a P450 fusion with a reductase domain that resembles phthalate dioxygenase reductase (PDOR). The purification of the intact CYP116B1 enzyme, and also of its isolated haem domain (expressed from the relevant gene section), is optimised and biophysical characterisations are reported. The haem iron redox potential is found to be unusually positive (-85 mV) and the influence of thiocarbamate herbicide substrate binding upon this potential is found to be minimal, unlike the case in CYP102A£ with its fatty acid substrates and likely as a consequence of the relatively small degree of shift in haem-iron spin-state towards the high-spin form. From a panel of eight potential substrates for CYP116B1, six were found to stimulate NADPH oxidation, but only two of these were themselves oxidised by the enzyme, with hydroxylated products observable. The genetically dissected reductase domain of CYP116B1 was also expressed and purified, and kinetic studies of the reductase domain revealed a preference for NADPH over NADH coenzyme, and enables comparisons with kinetic features and coenzyme selectivity in other members of the ferredoxin reductase family of enzymes. Collectively, these studies advance our knowledge of the properties of two distinct types of P450-redox partner fusion enzymes, a growing class of enzymes with potential for biotechnological applications.
23
Cirino, Patrick Carmen Arnold Frances Hamilton. "Laboratory evolution of cytochrome P450 peroxygenase activity /". Diss., Pasadena, Calif. : California Institute of Technology, 2004. http://resolver.caltech.edu/CaltechETD:etd-06062003-164310.
Testo completo
24
Fraser, David John 1968. "Isolation and characterization of cytochrome P450 3A26". Diss., The University of Arizona, 1998. http://hdl.handle.net/10150/288850.
Testo completoAbstract (sommario):
The objective of these studies was to isolate and characterize novel members of the 3A subfamily of canine cytochromes P450. This subfamily is of interest due to the wide range of endogenous and exogenous compounds metabolized. Previous studies led to the identification, isolation, and heterologous expression of 3A12, the major P450 form in canine liver. This enzyme demonstrated the ability to metabolize steroidal compounds and macrolide antibiotics in reconstituted systems. However, several lines of evidence suggested the presence of additional 3A enzymes with different substrate specificities. Initial experiments employed the same library used to clone the 3A12 cDNA and led to the isolation of a cDNA encoding P450 3A26. This new cDNA encoded a protein of 503 amino acids with 33 nucleotide differences encoding 22 amino acid variations compared with 3A12. Immunoblots indicated that 3A26 comigrates with a previously unidentified 3A protein in PB-induced canine microsomes. The 3A26 cDNA was modified for heterologous expression and cloned into the pSE380 expression vector. Expression of 3A26 and 3A12 in E. coli was accomplished using slightly modified protocols developed in this laboratory. Steroid hydroxylase assays indicated that these two enzymes have distinct catalytic profiles, with 3A12 exhibiting a rate of 6β-hydroxysteroid product formation ranging from 4 to 50-fold higher than 3A26. The vast differences in the activities of 3A12 and 3A26 in contrast to their similarity in structure made these enzymes an excellent model system for the identification of structure-function relationships. Studies were done to identify which of the 22 amino acid residue differences between 3A12 and 3A26 confer differences in rates of hydroxylation of progesterone, testosterone and androstenedione. Ten different 3A12/3A26 chimeras were generated using restriction endonuclease sites. Hydroxylase assays indicated that the first four residue changes and the six differences found within an internal PstI fragment were at least partially responsible for differences in the hydroxylation rates of all three steroids tested. Site-directed mutagenesis revealed the importance of 3A26 residues Ile-187, Ser-368, and Val-369. Conversion of these positions to 3A12 residues increased the rates of 6β-hydroxysteroid formation by 10-20 fold for progesterone, testosterone, and androstenedione. These studies identified a new member of the P450 3A subfamily and are the first to use catalytically distinct cytochromes P450 3A from the same species in the elucidation of 3A structure-function relationships.
25
Koenigs, Luke L. "Mechanism-based inactivation of P450 /". Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/8150.
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26
Perrin, Rachel. "Caractérisation de deux sous-familles d'isoenzymes du cytochrome p450 impliquées dans le métabolisme des xénobiotiques dans le cerveau". Nancy 1, 1991. http://www.theses.fr/1991NAN10462.
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27
Porro, Cristina Shino. "Quantum mechanical/molecular mechanics studies of Cytochrome P450BM3". Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/quantum-mechanical--molecular-mechanics-studies-of-cytochrome-p450bm3(ad4255e7-b779-47a2-a2c5-8dbf6b603ca5).html.
Testo completoAbstract (sommario):
Cytochrome P450 (P450) enzymes are found in all kingdoms of life, catalysing a wide range of biosynthetic and metabolic processes. They are, in fact, of particular interest in a variety of applications such as the design of agents for the inhibition of a particular P450 to combat pathogens or the engineering of enzymes to produce a particular activity. Bacterial P450BM3 is of particular interest as it is a self-sufficient multi-domain protein with high reaction rates and a primary structure and function similar to mammalian isoforms. It is an attractive enzyme to study due to its potential for engineering catalysts with fast reaction rates which selectively produce molecules of high value.In order to study this enzyme in detail and characterise intermediate species and reactions, the first step was to design a general hybrid quantum mechanical /molecular mechanics (QM/MM) computational method for their investigation. Two QM/MM approaches were developed and tested against existing experimental and theoretical data and were then applied to subsequent investigations.The dissociation of water from the water-bound resting state was scrutinised to determine the nature of the spin conversion that occurs during this transformation. A displacement of merely 0.5 Å from the starting state was found to trigger spin crossing, with no requirement for the presence of a substrate or large conformational changes in the enzyme.A detailed investigation of the sulfoxidation reaction was undertaken to establish the nature of the oxidant species. Both reactions involving Compound 0 (Cpd0) and Compound I (CpdI) confirmed a concerted pathway proceeding via a single-state reactivity mechanism. As the reaction involving Cpd0 was found to be unrealistically high, the reaction proceeds preferentially via the quartet state of CpdI. This QM/MM study revealed that the preferred spin-state and the transition state structure for sulfoxidation are influenced by the protein environment. P450cam and P450BM3 were found to have CpdI species with different Fe-S distances and spin density distributions, and the latter having a larger reaction barrier for sulfoxidation.A novel P450 species, the doubly-reduced pentacoordinated system, was characterised using gas-phase and QM/MM methods. It was discovered to have a heme radical coupled to two unpaired electrons on the iron centre, making it the only P450 species to have similar characteristics to CpdI. Calculated spectroscopic parameters may assist experimentalists in the identification of the elusive CpdI.
28
Liu, Kang-Cheng. "Influence of lipid membrane environment on the kinetics of the cytochrome P450 reductase- cytochrome P450 3A4 enzyme system in nanodiscs". Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/influence-of-lipid-membrane-environment-on-the-kinetics-of-the-cytochrome-p450-reductase-cytochrome-p450-3a4-enzyme-system-in-nanodiscs(b8ee4e84-1230-40cf-9b98-b5d6f457f54c).html.
Testo completoAbstract (sommario):
The cytochrome P450 enzyme system is a multicomponent electron-transfer chain composed of a haem-containing monooxygenase cytochrome P450 (CYP) and one or more redox partners. Eukaryotic CYPs and their redox partner NADPH-dependent cytochrome P450 oxidoreductase (CPR) are involved in many biological processes. Each protein has one N- terminal membrane anchor domain for location within the endoplasmic reticulum (ER). In mammals, CYPs and CPR are especially abundant in liver cells, where they play important roles in the metabolism of steroids, fatty acids, and xenobiotic compounds including numerous drugs of pharmaceutical importance. Incorporation into lipid membranes is an important aspect of CYP and CPR function, influencing their kinetic properties and interactions. In this thesis, soluble nanometer-scale phospholipid bilayer membrane discs, "nanodiscs", were used as a reconstitution system to study the influence of lipid membrane composition on the activities of the abundant human CYP3A4 and human CPR. Both enzymes were expressed and purified from bacteria, and assembled into functionally active membrane-bound complexes in nanodiscs. Nanodisc assembly was assessed by a combination of native and denaturing gel electrophoresis, and a fluorimetric assay was developed to study CYP3A4 reaction kinetics using 7-benzyloxyquinoline as substrate. Kinetic properties were investigated with respect to different lipid membrane compositions: phosphatidyl choline; a synthetic lipid mixture resembling the ER; and natural lipids extracted from liver microsomes. Full activity of the CYP3A4 system, with electron transfer from NADPH via CPR, could only be reconstituted when both CYP3A4 and CPR were membrane-bound within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated seperately into naodiscs then mixed together, or when soluble forms of CPR were mixed with pre-assembled CYP3A4-nanodiscs. Thus, assembly of the two proteins within the same membrane was shown to be essential for the function of the CPR-CYP3A4 electron transfer system. Comparison of the reaction kinetics in different membrane compositions revealed liver microsomal lipid to have an enhancing effect both on the activity of the assembled CPR-CYP3A4 nanodisc complex, and on the activity of CPR alone incorporated in nanodiscs, when compared either to the synthetic lipid mixture or to phosphatidyl choline alone. Thus, natural lipids appear to possess properties or include components important for the catalytic function of the CYP system, which are absent from synthetic lipid. Input of electrons, measured by NADPH consumption, exceeded product formation rate by the CPR-CYP3A4 complex in nanodiscs, indicating "leakage" in the electron flow, possibly due to uncoupling of the two enzymes. Uncoupling was shown to occur by developing a novel fluorimetric method using the dye MitSOX to detect superoxide production. The significance of this, and to what extent control of coupling could be a natural means of regulation of the CPR-CYP system, remains to be determined. Thus, phospholipid bilayer nanodiscs prove a powerful tool to enable detailed analysis of the reaction kinetics of membrane-reconstituted CPR-CYP systems, and to allow pertinent questions to be addressed concerning the integral significance of the membrane environment.
29
Diaz, Dominique. "Induction des cytochromes P450 par l'omeprazole : étude en culture primaire d'hépatocytes humains et résultats in vivo". Montpellier 1, 1989. http://www.theses.fr/1989MON11078.
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30
Marques-Soares, Cristina. "Topologie des sites actifs des cytochromes P450 2C8 et 2C9 de foie humain : étude de leur spécificité de substrats et utilisation de la structure RX du CYP 2C5 comme référence". Paris 11, 2002. http://www.theses.fr/2002PA112285.
Testo completoAbstract (sommario):
Pour étudier la topologie des sites actifs des CYP 2C9 et 2C8 humains, nous sommes partis de la première structure RX résolue d'un cytochrome P450 membranaire, le CYP 2C5 de lapin, fortement homologue aux CYP 2C humains. Nous avons tout d'abord construit un modèle 3D du CYP 2C9 par mutagenèse virtuelle du CYP 2C5. Ce travail ainsi qu'une étude de mutants du CYP 2C9 nous ont permis de souligner l'importance de certains résidus du CYP 2C9 dans sa reconnaissance et son oxydation des substrats. Dans un deuxième temps, nous avons élaboré de même un modèle 3D du CYP 2C8, tout en construisant indépendamment un pharmacophore de ses substrats. En confrontant les résultats de ces deux approches, nous avons proposé un modèle d'interaction du CYP 2C8 avec ses substrats. De plus le CYP 2C8 présent dans les vaisseaux sanguins semble être impliqué dans la régulation de la vasodilatation. En étudiant l'effet sur le CYP 2C8 de dérivés de voies impliquées dans la vasodilatation, les voies de la biosynthèse de NO et de la cascade arachidonique, nous avons identifié certains produits capables d'inhiber le CYP 2C8 in vitro. Enfin, nous avons trouvé des substrats et inhibiteurs communs aux CYP 2C5 et 2C humains, qui ont été cristallisés avec le CYP 2C5 par l'équipe de E. F. Johnson (La Jolla). Les structures RX obtenues ont permis de montrer une grande aptitude de l'enzyme à s'adapter a chacun de ses substrats. Ces résultats seront discutés en relation avec le comportement des cytochromes P450 2C humainsTo study the topology of the active site of human CYP 2C8 and 2C9 we used the first RX structure of a membrane cytochrome P450, the rabbit CYP 2C5, which presents a strong homology with the human CYP 2Cs. First, a 3D model of CYP 2C9 was constructed by virtual mutagenesis of CYP 2C5. This work, completed by a study of CYP 2C9 mutants, underlined some CYP 2C9 residues, important for the interaction with substrates and the catalytic efficiency of the enzyme. In a second step, a 3D model of CYP 2C8 was developed. Meanwhile a pharmacophore of CYP 2C8 substrates was constructed. By confronting the results of those two approaches we proposed a model of the interaction of these substrates with CYP 2C8. Moreover, CYP 2C8 seems to be implicated in the regulation of vasodilatation. By studying the effects on CYP 2C8 of derivatives from the other ways of control of vasodilatation (the way of NO biosynthesis and the arachidonic cascade), we identified a few products able to inhibit CYP 2C8. Finally, we found CYP 2C5 substrates and inhibitors common to human CYP 2Cs. Those molecules were crystallised with CYP 2C5 by the team of E. F. Johnson (La Jolla). The RX structures obtained showed that the enzyme adapts its structure to the substrate. By comparison, they also gave information on the human CYP 2Cs
31
Hukkanen, J. (Janne). "Xenobiotic-metabolizing cytochrome P450 enzymes in human lung". Doctoral thesis, University of Oulu, 2000. http://urn.fi/urn:isbn:9514258649.
Testo completoAbstract (sommario):
Abstract
The cytochrome P450 (CYP) enzyme system in human lung is an essential component
in the pulmonary carcinogenicity of several inhaled xenobiotic compounds. The
aim of this study was to elucidate the expression and regulation of
xenobiotic-metabolizing CYP enzymes in human lung.
To evaluate which of the two is a better surrogate cell model for lung tissue,
the expression patterns of CYP enzymes in alveolar macrophages and peripheral
blood lymphocytes were clarified by reverse transcriptase-polymerase chain
reaction (RT-PCR) and compared to the expression in lung tissue. The pattern of
CYP expression in alveolar macrophages was found to closely resemble the
expression pattern in human lung tissue, while the pattern in lymphocytes was
markedly different. The expression of CYP2B6, CYP2C, CYP3A5, and CYP4B1 mRNAs in
alveolar macrophages was demonstrated for the first time.
To facilitate mechanistic studies on human pulmonary CYP induction, the A549
lung adenocarcinoma cell line was characterized by RT-PCR, and the CYP
expression
pattern was found to compare reasonably well to human lung epithelial cells. The
induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) behaved as predicted, and CYP1B1 and CYP3A5 were also inducible by TCDD
and dexamethasone, respectively. TCDD elevated the level of CYP1A1 mRNA
(56-fold), while the induction of CYP1B1 mRNA was more modest (2.5-fold). The
tyrosine kinase inhibitor genistein and the protein kinase C inhibitor
staurosporine blocked CYP1A1 induction by TCDD, but did not affect CYP1B1
induction. The serine/threonine protein phosphatase inhibitor calyculin A and
okadaic acid enhanced CYP1B1 induction slightly, but did not alter CYP1A1
induction.
The expression of CYP3A forms in human pulmonary tissues was studied with RT-PCR
and
immunohistochemistry, and both methods established CYP3A5 as the main CYP3A
form. CYP3A4 was expressed in only about 20% of the cases. In A549 cells, CYP3A5
was induced about 4-fold by the glucocorticoids budesonide, beclomethasone
dipropionate, and dexamethasone. Maximal induction was achieved by
concentrations as low as ~100 nM, suggesting that CYP3A5 could be induced
in vivo in patients using inhaled glucocorticoids. However,
there were no differences in CYP3A5 expression in alveolar macrophages in
current glucocorticoid users, ex-users, and non-users. Cigarette smoking had a
marked decreasing effect on CYP3A5 levels in alveolar macrophages. The presence
and possible induction of CYP3A5 by glucocorticoids in human lung could have
consequences for the maintenance of physiological steroid hormone balance in
lung and the individual susceptibility to lung cancer of patients using
glucocorticoids.
32
Westlind, Johnsson Anna. "Pharmacogenetics of human cytochrome P450 3A (CYP3A) enzymes /". Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-688-x.
Testo completo
33
Hu, Yin. "Genetic polymorphism and regulation of cytochrome P450 2E1 /". Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3690-0.
Testo completo
34
Dapkūnas, Justas. "Computational modeling of cytochrome P450-mediated drug metabolism". Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2011. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2011~D_20111003_114651-54627.
Testo completoAbstract (sommario):
The main objective of this study was the development of QSAR models for drug metabolism-related properties. Novel GALAS (Global, Adjusted Locally According to Similarity) modeling method was used, which is a combination of baseline global QSAR model and local similarity based corrections. GALAS modeling method allows forecasting the reliability of prediction thus defining the model applicability domain. Models predicting CYP3A4 inhibition and regioselectivity of metabolism in human liver microsomes were developed and validated using external test sets. In all cases the baseline models already showed acceptable results, and the overall accuracy of predictions increased after the similarity based corrections. Moreover, the numbers of mispredictions reduced significantly when only results of higher reliability were taken into account. However, the original models are applicable only for less than a half of external datasets. Since the similarity correction procedure of GALAS modeling method allows simple model training, the possibility to expand the applicability domain has been tested. The CYP3A4 inhibition model was successfully adapted to PubChem data and compounds with a novel chemical scaffold. After training the regioselectivity model new metabolism sites could be identified in compounds of new chemical class. Moreover, this model was adapted for human cytochrome P450 isoform profiling.Pagrindinis šio darbo tikslas buvo kiekybinio struktūros ir aktyvumo ryšio modelių, prognozuojančių su vaistų metabolizmu susijusias savybes, kūrimas. Modeliai, prognozuojantys CYP3A4 slopinimą ir žmogaus kepenų mikrosomų katalizuojamo metabolizmo regioselektyvumą, buvo sukurti naudojant GALAS (angl. Global, Adjusted Locally According to Similarity; Globalus, lokaliai pakoreguotas pagal panašumą) modeliavimo metodą, kuris geba įvertinti prognozės patikimumą, taip apibrėždamas modelio pritaikymo sritį. Sukurtų modelių prognozės buvo tikrinamos naudojant eksperimentinius naujų cheminių junginių duomenis. Visų globalių modelių prognozės gerėjo po korekcijų pagal panašumą, o neteisingų spėjimų skaičius buvo ženkliai mažesnis tarp aukšto patikimumo prognozių. Visgi daugiau nei pusė išorinių duomenų nepatenka į šių modelių pritaikymo sritį. GALAS modeliai gali būti gana paprastai apmokomi, pridedant naujus duomenis į lokalią modelio dalį ir apskaičiuojant reikiamą korekciją. Po tokios apmokymo procedūros CYP3A4 slopinimo modelis prisitaikė prie PubChem duomenų bazės cheminių junginių ir taip pat prie vaistų, turinčių naują cheminį karkasą. Pridėjus naujų junginių ir apmokius regioselektyvumo modelį, jis pradėjo prognozuoti naujas metabolizmo vietas. Pastarasis modelis taip pat buvo pritaikytas atskirų fermentų katalizuojamo metabolizmo prognozavimui.
35
Harvey, Joanna Louise. "The induction of cytochrome P450 1A1 by metyrapone". Thesis, Queen Mary, University of London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300578.
Testo completo
36
Fairhead, Michael James. "Construction of human/bacterial cytochrome P450 fusion proteins". Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408140.
Testo completo
37
Fan, Ming Qi. "Studies of the structure of cytochrome P450 4A1". Thesis, University of Nottingham, 2002. http://eprints.nottingham.ac.uk/10400/.
Testo completoAbstract (sommario):
Cytochrome P450 4A1 (CYP4A1) is involved in omega-hydroxylation of fatty acids and eicosanoids. The resulting metabolites may have physiological activities such as regulation of blood pressure. In order to identify structural determinants of substrate binding, site-directed mutagenesis was used. According to a model of CYP4A1, the residues K93, R87 and N116 were predicted to respond to substrate binding. To test the hypothesis, we designed a series of mutants, K93E, R87E, R87E/K93E, R87W/K93E, N116E and N116E/K93E, which would change the substrate specificity of CYP4A1 from a fatty acid to a fatty amine omega-hydroxylase. To reconstitute CYP4A1 activity in vitro, cytochrome b5 and cytochrome P450 reductase were expressed in E.coli and purified. Recombinant CYP4A1 and mutants were expressed in E.coli with an OmpA signal peptide. The conditions of expression were optimised; the enzyme was purified by Ni2+-chelate affinity chromatography. Under optimal conditions, the expression level of CYP4A1 was approximately 60-100nmol/l; mutants were expressed at various levels. The purified enzymes were used in a spectral substrate-binding assay. The K93E mutation did not induce a major change in the substrate specificity from fatty acid to amine; however, K93E showed weak binding to dodecyltrimethylammonium bromide and the Ks (Spectral dissociation constant) value for binding lauric acid increased about three times. R87E mutants had low affinity for lauric acid, but did not show increased affinity for dodecyltrimethylammonium bromide. N116E is similar to wild type CYP4A1 in substrate affinity and specificity. N116/K93E could not be examined owing to the low expression level. These results suggest that K93 is not the principle residue for substrate binding but could be involved in transient contact with substrate; that R87 is crucial for keeping the substrate binding but might not directly contribute to binding of substrate and that N116 does not contribute directly to substrate contact. The residues, H141, R142, R143 and F149, which are located in the conserved Chelix in CYP4A1, were also investigated. We hypothesised that H141 and F149 bind to conserved residues in the I-helix. R142 and R143 may be involved in contacts with electron donors of CYP. Seven mutants including H141R, H141F, H141L, R142A, R143A, F149I and F149Y were constructed. All mutants were expressed and purified as for CYP4A1. R142A was expressed in low level and not further purified. F149I yielded proteins of the expected size, but these proteins did not support a 450nm peak in a reduced CO-difference spectrum, demonstrating an improperly folded enzyme. The enzyme activity of other mutants for lauric acid metabolism is variable; preliminary data showed that the H141L, H141F, R143A and F149Y had very poor enzyme activity, whereas the H141R retained enzyme activity. The results suggest that certain C-helix: I-helix contacts are not required for correct folding of the haem-environment, but are required for function of the P450 enzyme.
38
Caprotti, Domenico. "Control of electron transfer in cytochrome p450 enzymes". Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509901.
Testo completo
39
Tan, Hoon Leong. "Selective inhibitors of the cytochrome p450 enzyme CYP1B1". Thesis, De Montfort University, 2006. http://hdl.handle.net/2086/4295.
Testo completoAbstract (sommario):
The cytochrome P450 CYP1 ezymes, CYP1A1, CYP1A2 and CYP1B1, are members of the cytochrome P450 superfamily which catalyse the oxidative metabolism of a wide range of endogenous and exogenous compounds. CYP1B1 is highly overexpressed in different malignancies but not in the corresponding normal tissues. This significant discovery has provided an opportunity to develop tumour specific intracellular activated anticancer prodrugs, using CYP1B1 as molecular target. As part of the continuing CYP1B1 activated anticancer prodrugs discovery programme at Leicester School of Pharmacy, this research was set up to delineate the structure-activity relationship of the CYP1 enzymes. This was achieved by studying a range of inhibitors designed and synthesised during this Ph. D. project. The inhibitor's ability to inhibit CYP1 enzymes was quantified using a fluorometric high throughput ethoxyresorufin O-deethylase assay. DMU968 and DMU2157 were identified as inhibitors of CYP1A1. These inhibitors have low intrinsic toxicity and therefore, have potential applications for in vivo and cell line based in vitro experiments. 9-Acetylphenanthrene was identified as CYP1A2 inhibitor. It was demonstrated that 9-acetylphenahthrene has better potency and selectivity profiles compared with the known CYP1A2 inhibitor furafylline. Fourteen CYP1B1 inhibitors were identified. DMU778 and DMU2103 may have potential applications in cell based assays due to low intrinsic toxicity. DMU2123 and DMU2127 have been shown to possess tumour specific anticancer properties. These compounds were selectively activated by CYP1A1 and may have the potential as anticancer prodrug since some cancers also highly expressed CYP1A1. It was also found that residual insect P450, present in control microsomes, also bioactivated these compounds. Although the identity of the insect P450 has not been identified, DMU2123 and DMU2127 have a double potential as insect selective pesticides as well as tumour selective anticancer agents. Currently, more detailed studies are being performed on these compounds. Combining drug metabolism data obtained elsewhere and enzyme inhibition results, pharmacophore models for the CYP1 enzymes were constructed. The pharmacophore models for each CYP1 enzymes have shown distinct structural requirements for selective inhibitors and substrates. These pharmacophore models have contributed towards better prodrug design. Inhibitors synthesised in this research may be used for studying other P450s structure-activity relationships. Selective inhibitors identified in this project also provided valuable molecular probes for drug metabolism and pharmacokinetic studies.
40
Ridd, Thomas Ian. "Reactions and ligand binding properties of cytochrome P450". Thesis, University of Surrey, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308553.
Testo completo
41
Corcionivoschi, Nicolae. "A novel cytochrome P450 from Campylobacter jejuni 11168". Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/13463.
Testo completoAbstract (sommario):
Campylobacter jejuni is the most commonly recognized cause of bacterial gastroenteritis in man and also infects cattle, sheep and poultry. Publishing of the genome sequence of Campylobacter jejuni 11168 (Parkhill, 2000) revealed the presence of only one cytochrome P450. Its coding sequence (Cj411c) is located in an operon involved in sugar and cell surface biosynthesis. The gene name is Cj1411c, is 1359 bp long and encodes 453 aa. The sequence is strictly conserved in Campylobacter jejuni RM221. Recombinant P450 was expressed in E. coli and showed the 450 nm peak in the presence of CO indicating the correct folding. The protein was partially purified to about 70% purity. By deleting the P450 gene from the Campylobacter jejuni 11168 genome clear changes in cell morphology were identified, cells becoming wider and shorter. The capsular sugar profile of the NC1 knockout strain reveals the presence of arabinose which was not found in the wild type strain. The arabinose was identified by both HPLC and NMR. The phenotype studies showed clear differences between NC1 and WT cells: NC1 cells are less resistant to starvation in the stationary phase; by exposure to the atmospheric oxygen 36.47% of the wild type cells survived after 24 hours and only 16.61% of the NC1 cells survived; by growing the NC1 cells in competition with the WT cells the growth rate of NC1 cells approximately 10x lower than the WT; NC1 cells were proved to be less resistant to high temperature, more resistant to low temperatures and pH. By analysing the results obtained with NaC1 and glycerol we have determined that it is not the osmotic pressure that is affecting the growth of Campylobacter and the differences between the WT and NC1 strain might be related with changes in cell surface components.
42
Joyce, Michael Gordon. "Structural studies of cytochrome P450 BM3 and CprK". Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/29702.
Testo completoAbstract (sommario):
This work presents the crystal structure determination of the transcriptional regulator CprK and of individual domains of the multidomain cytochrome P450-BM3. The crystal structure of the A264E mutant heme domain was determined with and without substrate present. Surprisingly, the structures reveal the protein to exhibit a substrate bound conformation regardless of the presence of substrate. This has provided further evidence that substrate binding leads to a dramatic shift in the equilibrium of conformational states available to the protein. In addition, the crystal structure of the C773A mutant flavin binding domain has been determined both in presence and absence of NADP+. Together with the already available structures of the other domains, this now allows both modelling and further solution studies of the full length cytochrome P450-BM3 structure.;Dehalogenans sp. are capable of using a range of chlorophenolic compounds as terminal electron acceptors in a respiratory metabolism known as halorespiration. This process is under transcriptional control by CprK, a member of the CRP-FNR family of transcriptional regulators. The crystal structure of D. hafniense CprK in complex with o-chlorophenolacetic acid (OCPA) reveals tightly bound effector molecules. Binding of OCPA is analysed through both mutagenesis and fluorescence quenching binding studies. The results have led to the hypothesis that CprK uses the bound phenolic compound pKa as an additional mechanism to sense the presence of the chloride atom. The structure presents a structural framework for further studies of the mechanism of this family of transcriptional regulators and of CprK homologues in particular.
43
Urata, Kouji. "Hydrogen-driven hydrocarbon oxidation by cytochrome P450 enzymes". Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:d5ec728a-2aa8-4040-92b0-56dad59e6dc4.
Testo completoAbstract (sommario):
P450BM3 (CYP102A1) from Bacillus megaterium is a 119 kDa, 1,046-residue polypeptide with the FAD/FMN reductase domain fused to the C-terminus of the haem domain and, as such it is catalytically self-sufficient; only NADPH and oxygen are required for monooxygenase activity. P450BM3 is a sub-terminal fatty acid hydroxylase, but generations of CYP102A1 engineering allowed them to be used in, e.g. drug metabolism and alkane oxidation. This thesis describes protein engineering of P450BM3 and altering reaction conditions to enhance C1 â C8 alkane oxidation activities, with the long-term goal of oxidising methane, and using the hydrogen-driven cofactor regeneration system to drive these reactions. Catalytic hydrocarbon oxidation under mild conditions is highly desirable in fuel synthesis and energy applications. Methane is a greenhouse gas and its effect can be minimised if methane is selectively oxidised to methanol, which can be used as a liquid fuel or feedstock. The R47L/Y51F, KT2, I401P and A330P mutants, which previously showed higher activities for a wide range of substrates, were used as templates to build a library of mutants. The R47L/Y51F/KT2/A330P mutant (RT2/AP) showed total turnover number (TTN) of 680 ± 10 under atmospheric pressure at ambient temperature for propane oxidation, and the TTN improved by 16-fold under 5 bar propane pressure (TTN is defined as the maximum number of moles of substrate converted per mole of P450BM3). TTN values of 14,250 ± 1,370 (KU4/AP) and 920 ± 50 (KU3/AP) were observed under 5 bar propane and 8 bar ethane, respectively, at ambient temperature. The effect of adding an inert perfluorocarboxylic acid (PFC), which resembles the structures of natural substrates and constrains small alkanes to bind closer to the haem, was investigated. The R47L/Y51F/N70S/M237L/A328V mutant (RL/YF/NMA) with PFC11 gave a TTN of 13,590 ± 30 under 5 bar propane at ambient temperature. Higher TTNs of 26,320 ± 1,010 for propane and 1,440 ± 70 for ethane oxidation were observed for the RL/YF/NMA mutant at 4 °C due to improved aqueous alkane solubility. Octane and propane oxidations were performed using a hydrogen-driven NADP+ recycling system without changing the selectivity of products, although the observed propane oxidation activity was 15% of the glucose/GDH cofactor recycling system.
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Razalan, Maria Magdalena. "Mining the Arabidopsis genome for cytochrome P450 biocatalysts". Thesis, University of York, 2016. http://etheses.whiterose.ac.uk/16839/.
Testo completoAbstract (sommario):
Cytochromes P450 (CYPs) constitute a wide group of NAD(P)H-dependent monooxygenases, found throughout all kingdoms of life. Among the most important functions of CYPs are the synthesis of bioactive compounds and the conversion of xenobiotics. These functions can be translated into biotechnological applications, such as the production of highly regio- and stereo-specific drug metabolites for the pharmaceutical industry, or to confer activity towards toxic compounds for agronomics and bioremediation purposes. Plants possess a large number of CYP sequences, but most still remain uncharacterised, due to the difficulty in the isolation of the membrane-bound enzyme and in the reconstitution of an active and efficient redox system. In this project, a fusion construct for the co-expression of the CYP with a suitable reductase was created. The construct consisted of a C-terminal Arabidopsis ATR2 reductase (codon-optimised for expression in E. coli and truncated of the N-terminal membrane anchor) connected through a poly-GlySer linker to the heme domain. An N-terminal Im9 peptide replaced the natural membrane-binding domain of the CYP. When CYP73A5 from Arabidopsis was cloned into the construct, it was able to convert almost 60 % of the substrate cinnamic acid to the hydroxylated derivative, in whole cell assays. This result demonstrated that this expression platform enables the expression of active redox self-sufficient P450 catalysts and it can be further utilised for the characterisation of orphan CYPs. Following from gene expression studies and reports on the existence of oxidative derivatives of TNT, the potential involvement of CYP81D11 in the detoxification of TNT was explored with different in planta assays, employing transgenic Arabidopsis lines and tobacco leaf discs. The results obtained were contrasting and did not provide a clear picture on the role of CYP81D11. Further studies have to be carried out in the future, using CYP81D11-knockout lines, as well as the purified enzyme.
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Leishman, Amy G. "Cytochrome P450, CYP3 in the human fetal liver". Thesis, University of Aberdeen, 1991. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU045930.
Testo completoAbstract (sommario):
Previous work (Barnes et al, 1989) suggested that a different major im-munorelated form of CYP3 existed in adult and fetal human liver. Northern blot analysis was conducted, therefore, using an adult CYP3 cDNA probe, to determine how similar the fetal CYP3 messenger RNA species was to the adult. Two major messenger RNA species of similar length (2.1kb and 3.0kb) in both the adult and fetal liver were observed, indicating a high degree of homology between the two. A human fetal cDNA expression library in Agtll was prepared and screened using both an adult cDNA probe and monoclonal antibodies raised against a human adult liver CYP3, cytochrome P450 form (Barnes et al., 1987). This resulted in the isolation of a number of cDNA clones, one which was similar to (90% nucleotide sequence homology) but distinct from the adult cDNA sequences in the C7P3 gene family. Therefore, we have isolated and sequenced a partial cDNA cone which is unique to the human fetus and it appears that a different immunorelated form of C7P3 does exist in adult and fetal liver, with a distinct protein being expressed in the fetus. It is thought that one of the main physiological roles of this CFP3 protein in the fetus, is its involvement in production of estriol, required for the maintenance of pregnancy. In order to gain some insight into which specific amino acid changes, between the fetal and adult CYP3 proteins, might contribute to the altered substrate specificities observed in the CYP3 enzyme (Cresteil et al., 1982; Kitada et al., 1987), the positions of these changes were analyzed with respect to the tertiary structure of P450cam. This allowed speculation concerning which specific amino acid changes may be important in affecting the metabolic profile of the CYP3 protein and furthermore, the amino acids that may be important in the substrate access channel. On comparison of protein CYP3 levels in both adult and fetal livers (Western blot analysis) with clinical data, the OT3 protein expression was noted to be higher in an adult patient treated with an anticonvulsant drug, than in patients receiving no drug treatments. Furthermore, large interindividual differences in CYP3 protein expression levels were seen in adult livers, although this was not as pronounced in fetal livers. This perhaps reflects the need for more constant levels of the CYP3 protein in all human fetuses, due to the vital role it plays in the production of estriol.
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Kelly, Paul. "Engineering cytochrome P450-reductase fusion enzymes for biocatalysis". Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/engineering-cytochrome-p450reductase-fusion-enzymes-for-biocatalysis(c4b3aa48-1c73-4980-b480-e3a881267ee5).html.
Testo completoAbstract (sommario):
Cytochromes P450 (P450s) are a superfamily of heme-thiolate monooxygenases. They catalyse a wide variety of reactions on a vast number of substrates and are of particular interest for biocatalyst development due to their ability to oxidise non-activated C-H bonds. Fusion of a P450 to a suitable redox partner protein produces a catalytically self-sufficient enzyme and removes the need to produce electron transfer proteins separately. The well-studied bacterial protein P450cam (Pseudomonas putida) has been fused to the reductase (RhFRed) from the natural fusion protein P450-RhF (Rhodococcus sp.). The P450cam-RhFRed system catalyses the oxidation of camphor and several non-natural substrates and served as the basis for P450cam re-engineering in this current project, with the aim of expanding the substrate scope towards a more mammalian-like activity. The P450cam active site was partitioned into seven paired amino acids and each pair randomised in turn to generate seven sub-libraries of P450cam variants. These were screened for activity using a specially developed colony screen for detection of the blue pigment indigo. In total 94 new variants were identified and then pooled for secondary screening on a number of new substrates, identifying potentially novel activities within the ‘indigo positive’ population. In a separate ‘chimeragenesis’ approach substrate recognition sites (SRSs) within P450cam were targeted for exchange with equivalent portions from a number of human P450s. The B’ helix and F-G loop regions from CYPs 1A2, 2C8, 2D6 and 3A4 were grafted onto the P450cam structure and several of the B’ helix swaps were produced as soluble proteins. The P450cam-2C8-B’-RhFRed chimera gave a Soret peak at 420 nm in the Fe(II)-CO state although an additional substitution next to the proximal cysteine appeared to restore a P450-like state. SRS-exchange therefore offered some insight into structural modularity in P450s, providing a basis for further biocatalyst development.
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Aoyama, Ronald Gordon. "Probing the active site of cytochrome P450 CYP2C9 /". Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8188.
Testo completo
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Quesnot, Nicolas. "Évaluation de la génotoxicité des contaminants environnementaux, production de lignées bio-senseurs et mesure de l'activité enzymatique du cytochrome P450 2E1 dans les cellules d'hépatome humain HepaRG". Thesis, Rennes 1, 2015. http://www.theses.fr/2015REN1B005/document.
Testo completoAbstract (sommario):
L'exposition humaine aux contaminants environnementaux est inévitable du fait de leur présence dans l'eau, l'air et l'alimentation. La plupart d'entre eux sont reconnus comme étant mutagènes et/ou carcinogènes chez l'animal mais ils sont souvent seulement suspectés de l'être chez l'Homme. Le manque de connaissance vis-à-vis des substances chimiques a conduit l'UE à lancer le programme REACH avec l'objectif d'évaluer la toxicité d'environ 30 000 molécules. Cette évaluation nécessiterait l'utilisation de plus de 4 millions d'animaux et la pertinence controversée de ces modèles pourrait aboutir à des conclusions discutables. Les méthodes in vitro sont considérées comme une alternative potentielle à l'expérimentation animale. Néanmoins, le choix du modèle cellulaire et des conditions expérimentales restent à préciser. Les hépatocytes humains en culture primaire représentent le modèle le plus pertinent en toxicologie malgré de nombreuses contraintes (variabilité inter-individuelle, changements phénotypique précoces, obtention aléatoire). La lignée HepaRG constitue une alternative intéressante puisque ces cellules peuvent proliférer de manière illimitée et expriment les EMXs à des niveaux proches des hépatocytes humains. L'expression de ces enzymes restant stable pendant plusieurs semaines, ce modèle permet l'évaluation du risque lié à une exposition chronique aux contaminents environnementaux, essentielle en génotoxicité. Il reste cependant nécessaire de caractériser plus amplement cette lignée vis-à-vis des EMXs et de l'adapter aux tests de toxicologie actuels. Dans ces travaux, nous avons développé un test haut débit utilisant la quantification in situ des histones phosphorylées γH2AX avec l'objectif de pouvoir évaluer le risque génotoxique d'une exposition unique ou répétée aux contaminants environnementaux. Ce test a été validé avec succès par l'évaluation de la génotoxicité associée à une exposition de 1, 7 et 14 jours pour 10 polluants. Nous avons ensuite généré des lignées recombinantes biosenseurs, dérivées du modèle HepaRG et permettant d'identifier les xénobiotiques altérant l'expression transcriptionnelle des EMXs. Par transfection transitoire, nous avons dans un premier temps validé à l'aide d'inducteurs prototypiques et de nos 10 contaminants nos constructions contenant le gène rapporteur de la luciférase sous le contrôle des promoteurs de plusieurs EMXs. Ensuite, nous avons généré des lignées stables exprimant la GFP comme gène rapporteur et permettant une détection rapide des xénobiotiques capables d'induire l'expression des EMXs. Parmi les EMXs, le CYP2E1 joue un rôle important en santé humaine. En effet, cette enzyme induite dans certaines conditions physiopathologiques comme le diabète et l'obésité est responsable de l'activation de nombreux procarcinogènes et est à l'origine d'une production d'EROs. Les cellules HepaRG pourraient constituer un modèle pertinent pour l'étude du CYP2E1. Cependant, l'expression et l'activité de cette enzyme au sein de ce modèle nécessitent d'être mieux caractérisées en regard des données discordantes de la littérature. A l'aide de la chlorzoxazone, un marqueur spécifique de l'activité du CYP2E1, nous avons démontré l'influence du métabolisme de phase II sur l'activité apparente du CYP2E1. Nous proposons ici quelques recommandations afin de mieux quantifier l'activité du CYP2E1 sur les hépatocytes humains et sur le modèle HepaRG à l'aide la chlorzoxazoneHuman exposure to toxic chemicals is virtually unavoidable due to contamination of air, water and food. A number of environmental contaminants are recognized as mutagenic and/or carcinogenic in animal but they are often only suspected to have similar effects in Humans. The lack of knowledge on the effects of most industrial-made chemicals has led the EU to launch the REACH program with the aim of evaluating the toxicity of more than 30.000 molecules. Such evaluation would require the use of at least 4 millions of animals for an estimated cost of 2.8 billions €. While the relevance of these in vivo models remains controversial
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Zhao, Jin 1975. "Investigations of the biocatalytic activity of human P450 2D6". Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111940.
Testo completoAbstract (sommario):
The cytochrome P450 enzymes (CYPs) are very attractive biocatalysts because of their ability to regio- and stereo-selectively catalyze the insertion of a single atom of molecular oxygen into inactivated C-H bonds. There are many drawbacks, however, limiting the use of these enzymes in organic synthesis, including the need for expensive cofactors, low stability, and low tolerance to organic solvents. The goal of this thesis was to overcome some of these drawbacks for human CYP2D6. This isoform was selected because of its broad substrate promiscuity and high importance in drug metabolism.We have tested inexpensive chemicals to replace the natural cofactors of CYP2D6, NADPH and cytochrome P450 reductase (CPR). The results showed that cumene hydroperoxide and tert-butyl hydroperoxide can successfully substitute CPR and NAD(P)H with retained regio- and stereo-selectivity. Moreover, with these surrogates, product formation and initial rates are increased by as much as two fold compared to the use of the natural cofactors.
It is widely accepted that even small proportions of organic solvents in the buffer can deactivate most enzymes including P450s. Our studies on the biocatalysis of CYP2D6 in organic solvent/buffer emulsions showed that under the optimized conditions, as much as 76% of the enzyme activity was retained. Product formation in biphasic solvent systems is comparable whether the natural redox partner and cofactor are used, or a surrogate. In addition, a correlation was observed between the log P and the suitability of a solvent for enzymatic activity, with higher log P resulting in higher enzymatic activity. These results were obtained with dextromethorphan (DXM), a water soluble substrate. A very hydrophobic substrate, 7-benzyloxy-4-N,N-diethylaminomethyl-coumarin (BDAC), was also tested successfully to demonstrate the utility of this method.
Lyophilization is usually required to remove water before using enzymes in nearly anhydrous solvents. This physical process is harmful to P450 enzyme activity. We therefore tested numerous sugars as lyoprotectant during lyophilization. Addition of trehalose or sucrose before lyophilization allowed the retention of 80% of the CYP2D6 activity, compared to 40% remaining activity in its absence. CYP2D6 co-lyophilized with trehalose was next tested in selected hydrophobic organic solvents in the absence of water. The enzymatic activity was found to strongly depend on the hydrophobicity of the solvent. Interestingly, the enzyme showed higher catalytic ability in n-decane or n-dodecane than in the standard buffer. This was unexpected considering that the activity of most enzymes was reported to decrease to 10% or less in nearly anhydrous organic solvents.
The last objective of this thesis was to improve the stability and/or activity of CYP2D6. Use of DNA self-assemblies to encapsulate P450 enzymes was envisaged to potentially increase their stability. Indeed, DNA assemblies have many advantages compared to traditional solid supports reported for enzymes. Our preliminary results showed that CYP2D6 templated the formation of cyclic DNA dimeric and tetrameric over polymeric self-assemblies. Characterization of the CYP2D6 activity in the presence of the DNA self-assemblies revealed no loss of activity or stability.
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Stahl, Gunther. "Entwicklung von Homologie-Modellen der Cytochrome P450 2A5 und 2A6". [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964988984.
Testo completo