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Vincent, Thierry. "Optimisation des conditions réactionnelles et création de nouveaux mutants à grande performance du cytochrome p450 BM3 CYP102A1 utilisant les cofacteurs alternatifs NADH et N-benzyl-1,4-dihydronicotinamide." Doctoral thesis, Université Laval, 2020. http://hdl.handle.net/20.500.11794/66678.
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Le cytochrome p450 CYP102A1, mieux connu sous le nom de BM3, provient de la bactérie Bacillus megaterium. Cette enzyme possède un groupement prosthétique hémique lui permettant de catalyser l’insertion d’oxygène dans un lien carbone-hydrogène menant généralement à une hydroxylation du substrat, ce qui en fait une monooxygénase. Ce genre de réaction demeure jusqu’à aujourd’hui difficile à effectuer par chimie traditionnelle ce qui confère un intérêt particulier à cette enzyme. Au contraire des autres cytochromes p450, BM3 est soluble (et non membranaire) et est naturellement fusionnée à son partenaire réductase formant ainsi une seule chaîne polypeptidique. Ainsi, au cours des dernières années, BM3 a attiré beaucoup d’attention de la part de l’industrie de la chimie fine et pharmaceutique due à son potentiel biocatalytique important. Cependant, son usage en industrie est restreint par son instabilité ainsi que par le coût prohibitif du cofacteur qui lui est nécessaire pour catalyser ses réactions, le NADPH. Cette thèse décrit le développement de différentes stratégies visant à libérer les réactions effectuées avec BM3 de leur dépendance au NADPH, tout en maximisant le rendement spécifique de la monooxygénase. En place du NADPH, deux autres cofacteurs de moindre coût furent utilisés comme alternative, soit le NADH et le N-benzyle-1,4- dihydronicotinamide (NBAH) en utilisant le mutant R966D/W1046S de BM3. Afin de maximiser le rendement spécifique de BM3, l’une des stratégies de cette thèse, l’optimisation du milieu réactionnel, repose sur deux éléments clés, soit favoriser la stabilité du cofacteur, car celui-ci est plus instable que l’enzyme elle-même, ainsi que d’abaisser au minimum la température de la réaction, car nous avons constaté que ceci avait pour effet d’augmenter le couplage entre les réactions réductase et monooxygénase et donc la stabilité de l’enzyme. L’effet net de la réaction ainsi optimisée fut d’augmenter le rendement spécifique du mutant R966D/W1046S par un facteur situé entre 2 et 2.6 en fonction du cofacteur utilisé. D’autre part, deux stratégies d’ingénierie enzymatique furent explorées afin de générer des mutations pouvant augmenter la performance de BM3. L’une d’entre elles, la mutagenèse par consensus guidé, généra une librairie de mutants de laquelle les mutants NTD5 et NTD6 furent identifiés, augmentant le rendement spécifique de l’enzyme comparativement à leur parent, R966D/W1046S, par un facteur de 5.2 et 2.3 pour le NBAH et le NADH, respectivement. L’autre stratégie explorée fut d’appliquer une pression sélective sur la bactérie Bacillus megaterium pour forcer, par évolution expérimentale, la performance de l’enzyme. De cette stratégie, un nouveau mutant de BM3 nommé DE, possédant 34 acides aminés substitués sur sa séquence, fut généré. Ce dernier a démontré une plus forte résistance aux solvants organiques ainsi qu’une augmentation de son rendement spécifique vis-à-vis le NADPH et le NADH d’un facteur de 1.23 et 1.76, comparativement à BM3 sauvage, respectivement. Les stratégies décrites dans cette thèse présentent une amélioration significative du rendement spécifique de BM3 ainsi que deux iii nouvelles méthodologies avec lesquelles une enzyme peut être optimisée et de nouvelles mutations bénéfiques identifiées.The p450 cytochrome CYP102A1, better known as BM3, comes from the bacteria Bacillus megaterium. This enzyme possesses a prosthetic heme group enabling it to catalyze the insertion of oxygen into a carbon-hydrogen bond generally resulting in the hydroxylation of the substrate, the enzyme is therefore a monooxygenase. This type of reaction remains difficult to achieve by traditional chemistry. Unlike other p450 cytochromes, BM3 is soluble (is not membrane bound) and is naturally fused to its reductase partner forming a single polypeptide chain. As such, in recent years, BM3 has garnered much attention from the pharmaceutical and fine chemical industries, due to its high biocatalytic potential. However, its use in industry remains constrained by its instability as well as by the prohibitive cost of its cofactor, NADPH. This thesis describes the development of different strategies aiming at liberating reactions driven with BM3 from their dependence to NADPH whilst maximizing the specific yield of the monooxygenase. Instead of NADPH, two other inexpensive cofactors were used, namely NADH and N-benzyl-1,4-dihydronicotinamide (NBAH) by using the BM3 mutant R966D/W1046S. To maximize BM3 specific yield, one of the strategies used in this thesis work, the optimization of the reaction medium, rested on two key elements. Firstly, favouring the stabilization of the cofactor, as it was found to be more unstable than the enzyme itself and secondly lowering the reaction temperature as this effectively augmented oxidase/reductase reactions coupling and as such the stability of the enzyme. The net effect of the optimized reaction was to enhance the specific yield of the BM3 mutant R966D/W1046S by a factor of 2 and 2,6 depending on which cofactor was used. Two other enzymatic engineering strategies were explored to generate mutations which could enhance the performance of BM3. One of these, consensus guided mutagenesis, generated a library of mutants from which mutants NTD5 and NTD6 were identified enhancing the specific yield of the enzyme comparatively to their parent, R966D/W1046S, by a factor of 5,24 and 2,3 for NBAH and NADH respectively. The other strategy explored was to apply a selective pressure on Bacillus megaterium to force, by experimental evolution, the performance of the enzyme. From this strategy, a new mutant of BM3 called DE, possessing 34 new amino acid substitutions, was generated. This new mutant displayed a greater resistance to organic solvents as well as an augmentation of specific yields when used alongside NADPH and NADH comparatively to wild type BM3 by a factor of 1,23 and 1,76 respectively. The strategies described in this thesis allowed a significative enhancement of BM3 specific yield as well as represent two new methodologies by which new beneficial mutations can be identified.
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Trigui, Mohamed. "Biodégradation des amines cycliques par Mycobacterium sp. RP1 et Pseudomonas putida O1G3 : Caractérisation des monooxygénases impliquées dans l'hydroxylation des liaisons C-N." Compiègne, 2002. http://www.theses.fr/2002COMP1415.
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L’aptitude des microorganismes à dégrader les molécules synthétisées par l’industrie peut être mise à profit pour décontaminer des sites pollués. La morpholine (C4H9ON), la pyrrolidine (C4h9N) et la pipéridine (C5H11N) sont des amines secondaires très utilisées par l’industrie chimique. Ces amines peuvent subir diverses réactions chimiques et être transformées en N-nitosocomposés par action du NO2. Ces N-nitrosocomposés, même présents en faible concentration, ont un effet mutagène et cancérigène. En conséquence, la présence de ces polluants dans l’environnement présente un danger pour la santé publique. Ces amines sont biodégradables par des bactéries à Gram positif appartenant au genre Mycobacterium comme Mycobacterium sp. Rp1 qui est capable de les utiliser comme seule source de carbone, d’azote et d’énergie. L’utilisation de la métyrapone et des mesures spectrophotométriques ont permis de confirmer l’implication d’un cytochrome P450 dans la dégradation de ces amines cycliques. Nous avons associé des techniques de biologie moléculaire à des mesures de chromatographie d’affinité pour la purification de la protéine. Nous avons réalisé une construction génétique, permettant l’expression d’un cytochrome P450 recombinant et portant un motif His Tag en N-terminal pour une purification en une seule étape de la protéine (CYP151A2). Cette hémoprotéine présente un poids moléculaire de 46 kDa et montre un spectre d’absorption avec un maximum à 448 nm en présence de monoxyde de carbone. D’autre part, les paramètres montrent que cette monoxygénase présente une faible affinité pour les amines cycliques, et une vitesse de transformation de même ordre pour la pyrolidine et la morpholine mais moins importante pour la pipéridine. Une seconde partie de ce travail s’est focalisée sur l’étude de la dégradation des amines secondaires par Pseudomonas putida O1G3, bactérie à Gram négatif. Cette souche est capable de pousser sur milieu riche en pyrrodine et pipéridine mais pas en morpholine. Une caractérisation microbiologique et la détermination des différents paramètres cinétiques de dégradation de ces amines cycliques ont permis d’évaluer les capacités dégradatives de la souche O1G3. Différentes expériences comprenant la recherche des substrats de croissance, ont montré que la première étape de dégradation de ces amines est l’ouverture du cycle suite à la coupure de liaison C-N. Au vu de l’ensemble de ces résultats, nous avons proposé un modèle métabolique de la voie de dégradation de la pyrrolidine. Les analyses spectroscopiques et la recherche de l’enzyme impliquée dans l’ouverture des cycles des ces amines, ont montré l’implication d’une monooxygénase NADH dépendante. Cette monooxygénase présente une analogie avec la diméthylamine monooxygénase. La biodégradation des amines cycliques est possible par des bactéries Gram-positif via des monooxygénases de type amine monooxygénase P540 respectivement.
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Buronfosse, Thierry. "Métabolisme énantiosélectif de trois molécules à groupement thioéthers par des monooxygénases à cytochrome P450 et à flavine chez le rat : implications pharmacologiques et toxicologiques." Lyon 1, 1995. http://www.theses.fr/1995LYO1T202.
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Mascia, Francesco. "Engineering ferredoxin-dependent oxyfunctionalization in cyanobacteria." Electronic Thesis or Diss., Aix-Marseille, 2022. http://www.theses.fr/2022AIXM0648.
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Les cyanobactéries drainent une attention grandissante en tant que photo-biocatalyseurs répondant aux critères de la Chimie Verte. Elles sont capables de croître en utilisant uniquement la lumière et le CO2 comme sources d’énergie et de carbone. L’ajout de donneurs d’électrons sacrificiels (ie glucose) pour le recyclage du cofacteur NADPH d’oxydoréductases n’est pas nécessaire car celui-ci est régénéré par des électrons issus de l'oxydation photosynthétique de l'eau tandis que les oxygenases peuvent utiliser le dioxygène produit in-situ lors de la photosynthèse. Une souche de Synechocystis sp. PCC 6803, modifiée pour exprimer CYP153A6, un cytochrome P450, hydroxyle sélectivement le limonène, substrat bon marché et largement disponible, en l'alcool périllylique, utilisable comme arôme ou médicament. Une autre souche, exprimant le seul CYP110D1 et non les protéines porteuses d'électrons de ce cytochrome P450, catalyse l'hydroxylation regiosélective de la testostérone en 15β-hydroxytestostérone, davantage biodisponible et adapté aux formulations orales. L’activité (1 U gCDW-1) est deux fois plus grande que celle des réactions biocatalysées par la bactérie Escherichia coli. Une protéine de fusion CYP110D1- Fed1, une des ferrédoxines natives de Synechocystis, a également été conçue, visant à canaliser plus efficacement les électrons du photosystème I vers la monooxygénase.Ce travail a démontré l'efficacité des cyanobactéries modifiées exprimant des cytochromes P450 lorsqu’elles sont utilisés en tant que biocatalyseurs dans des procédés à cellules entières. Elles permettent la production durable de produits de grande valeur, tels que les produits pharmaceutiquesCyanobacteria are attracting growing attention as photo-biocatalysts meeting the criteria of Green Chemistry. They are able to grow using only light and CO2 as energy and carbon sources. The addition of sacrificial electron donors (i.e. glucose) for the recycling of the NADPH cofactor of oxidoreductases is not necessary because it is regenerated by electrons from the photosynthetic oxidation of water, while the oxygenases can use the oxygen produced in-situ during photosynthesis. A strain of Synechocystis sp. PCC 6803, modified to express CYP153A6, a cytochrome P450, selectively hydroxylates limonene, a cheap and widely available substrate, to perillyl alcohol, usable as a flavor or drug. Another strain, expressing only CYP110D1 without any electron-carrier proteins of this cytochrome P450, catalyzes the regioselective hydroxylation of testosterone to 15β-hydroxytestosterone, which is more bioavailable and suitable for oral formulations. The activity (1 U gCDW-1) is twice as high as that of the reactions biocatalyzed by the bacterium Escherichia coli. A CYP110D1-Fed1 fusion protein, one of the native Synechocystis ferredoxins, was also designed, aiming to channel photosystem I electrons more efficiently to monooxygenase. This work demonstrated the efficacy of modified cyanobacteria expressing cytochromes P450 when used as biocatalysts in whole-cell processes. They enable the sustainable production of high-value products, such as pharmaceuticals
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Mougin, Christian. "Métabolisme oxydatif du chlortoluron chez des cultures cellulaires de blé : intervention de monooxygénases à cytrochrome P-450." Toulouse, INPT, 1990. http://www.theses.fr/1990INPT034G.
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Les systemes enzymatiques responsables du metabolisme oxydatif d'un herbicide, le chlortoluron, ont ete etudies dans des microsomes isoles de cultures cellulaires de ble, qui renferment egalement de bons niveaux d'activites cinnamate 4-hydroxylase et laurate hydroxylase. Le chlortoluron est oxyde en un derive hydroxyle sur le methyle du cycle aromatique et en un derive n-monodemethyle. Les deux activites enzymatiques possedent les proprietes caracteristiques des monoxygenases a cytochromes p-450 (localisation cellulaire, besoins en co-facteurs, sensibilite a des inhibiteurs connus de monooxygenases a cytochrome p-450). Les quatre activites mesurees (cinnamate 4-hydroxylase, laurate hydroxylase, hydroxylase n-demethylase du chlortoluron) et les teneurs en cytochrome p-450 sont augmentees apres des traitements des cellules par des composes varies (herbicides, fongicides et antidotes d'herbicides). Des etudes qsar ont montre que des analogues structuraux du chlortoluron modifient le metabolisme de celui-ci. Prises dans leur ensemble, ces donnees suggerent que des monooxygenases a cytochrome p-450 sont impliquees dans les reactions d'hydroxylation et de n-demethylation de l'herbicide. Les caracteristiques des deux activites laissent penser que des enzymes distinctes catalysent chacune des deux reactions
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Lemoine, Antoinette. "Rôle des monooxygénases à cytochrome P-450 et à flavine dans le métabolisme de l'imipramine : étude de la régulation hormonale chez le rat." Paris 11, 1991. http://www.theses.fr/1991PA114837.
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Clair, Philippe. "Expression de cytochromes P-450, de la NADPH cytochrome P-450 réductase et de monooxygénases à flavine par baculovirus recombinants : Une contribution à l'étude du métabolisme des xénobiotiques chez l'homme." Montpellier 2, 1993. http://www.theses.fr/1993MON20248.
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Les produits de plus de 25 genes cytochrome p450 (cyp) et 5 genes flavine monooxygenase (fmo) sont exprimes chez l'homme, et sont responsables du metabolisme de phase i de la plupart des medicaments et xenobiotiques. L'expression par genie genetique d'enzymes humaines clonees repond specifiquement aux problemes d'obtention de materiel enzymatique pur, abondant, et actif necessaire a l'etude du metabolisme des xenobiotiques. Des baculovirus recombinants contenant les cdnas des enzymes cyp3a4, cyp2a6, cyp27, cyp3a6, fmo1, fmo3, fmo4, cytochrome p450 reductase (ncpr) et une cytochrome p450 reductase soluble ont ete construits et les enzymes produites ont ete caracterisees quant a leur taille, leur localisation sub-cellulaire, leur spectre d'absorption et leurs activites enzymatiques en systeme reconstitue. Une optimisation du systeme est decrite qui permet l'incorporation d'heme par l'apoenzyme cyp. Les mesures d'activite cyp2a6 demontrent la labilite de la reductase endogene des cellules hotes sf9 et la necessite de complementer les microsomes sf9 par la ncpr. L'activite des enzymes fmo est etudiee sur plusieurs substrats, ainsi que la stereoselectivite des fmo1 et fmo3 vis-a-vis d'un substrat prochiral. Le systeme baculovirus/sf9 est convenable pour la production d'enzymes fmo et ncpr, et, moyennant quelques adaptations specifiques, pour la production d'enzymes cyp
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Siddique, Muhammad Hussnain. "Study of the biosynthesis pathway of the geosmin in Penicillium expansum." Thesis, Toulouse, INPT, 2012. http://www.theses.fr/2012INPT0085/document.
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La géosmine est un terpénoïde, provoquant un goût moisi-terreux associée à des flaveurs atypiques dans l'eau et le vin. Chez les bactéries, la voie de biosynthèse de la géosmine est bien caractérisée, mais peu de connaissance sont disponibles au sujet de sa biosynthèse chez les eucaryotes, en particulier dans les champignons filamenteux. L'origine de la géosmine dans la vigne est en grande partie attribuable à la présence de Penicillium expansum sur les raisins. Dans cette thèse, afin de mieux comprendre la voie de biosynthèse de la géosmine chez Penicillium expansum, nous avons décrit la caractérisation et l'analyse de "gpe1", un gène codant pour une cytochrome P450 monooxygénase impliquée dans la biosynthèse de la géosmine. Nous avons démontré que les deux fragments d'ADN: p450-1 et p450-2 appartiennent à un seul gène du cytochrome p450 (gpe1). La séquence d'acides aminés déduite de gpe1 a une identité moyenne de 40 % avec les enzymes PbP450-2 et P450-4 qui ont été trouvées impliquées respectivement dans la synthèse d'indole diterpène et dans la synthèse des gibbérellines. Les amplifications par PCR effectuée sur quatorze espèces de Penicillium ont montré que seules les espèces producteurices de la géosmine ont donné le même fragment de ~1,2 kb que gpe1. L'analyse du gène gpe1 nous a permis d'identifier la présence de certains domaines conservés de cytochromes P450 monooxygénases. Ensuite, la caractérisation fonctionnelle du gène gpe1 chez P. expansum M2230 a été décrite. Nous avons montré que les mutants de gpe1 ont perdus leur pouvoir de produire la géosmine alors que les révertants de gpe1 ont rétablis leur pouvoir de production. Enfin, nous avons démontré qu'une polykétide synthase putative et une putative NRPS sont présentes sur le côté droit du gène gpe1 proposant que le gène gpe1 pourrait être une partie d'un «Cluster» codant pour la biosynthèse de métabolites secondairesGeosmin is a terpenoid, an earthy-musty compound associated with off-flavors in water and wine. In bacteria, the biosynthesis pathway of geosmin is well characterized, but little is known about its biosynthesis in eukaryotes, especially in filamentous fungi. The origin of geosmin in grapevine is largely attributable to the presence of Penicillium expansum on grapes. In this thesis, we have described the characterization and analysis of "gpe1", a gene encoding a cytochrome P450 monooxygenase probably involved in the biosynthesis of geosmin in P. expansum M2230, in order to better understand of the biosynthesis pathway of geosmin in this species. We demonstrated that the two DNA fragments i.e. p450-1 and p450-2 belong to a single cytochrome p450 gene (gpe1). We showed that the deduced amino acid sequence of gpe1 has an average identity of 40 % with PbP450-2 and P450-4 enzymes which have been found involved in indole diterpene synthesis and in gibberellin synthesis respectively. Then, the results of PCRs performed on the fourteen Penicillium species showed that only Penicillium species which were producers of geosmin gave the same fragment of ~1.2 kb like gpe1. Analysis of the gpe1 gene enabled us to identify the presence of some conserved domains of cytochromes P450 monoxygenases in the amino acid sequence of gpe1. Then, the functional characterization of the gpe1 gene in P. expansum M2230 was described. We illustrated that the mutants of gpe1 lost their potential to produce geosmin whereas the reverse complements of gpe1 restored their potential to produce geosmin. Finally, we demonstrated that a putative polyketide synthase and a putative NRPS-like enzyme are present on the right side of the gpe1 gene suggesting that gpe1 gene might be the part of a gene cluster encoding the biosynthesis of secondary metabolites
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Lafite, Pierre. "ETUDE du CYTOCHROME P450 2J2 HUMAIN :Recherche de substrats et d'inhibiteurs sélectifs ;Détermination de la topologie de son site actif." Phd thesis, Université René Descartes - Paris V, 2007. http://tel.archives-ouvertes.fr/tel-00192090.
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Ce manuscrit présente une étude fonctionnelle et structurale du cytochrome P450 2J2 humain (CYP2J2), enzyme exprimée dans les tissus cardiovasculaires, dont les rôles biologiques sont mal connus. En utilisant la terfénadone comme base structurale, qui est un composé oxydé régiosélectivement par le CYP2J2, plusieurs composés ont été synthétisés se sont révélés être des inhibiteurs affins et sélectifs du CYP2J2. En particulier, un inhibiteur très affin et compétitif (Ki = 160 nM) et deux substrats suicides efficaces du CYP2J2 (kinact/Ki 3000 L/mol/s) ont été mis en évidence. L'étude de l'oxydation de ces composés par le CYP2J2 a révélé une régiosélectivité surprenante, en faveur d'une position chimiquement moins réactive vis-à-vis des oxydations. La caractérisation du site actif du CYP2J2 et l'identification de résidus importants pour la reconnaissance des dérivés de terfénadone a pu être réalisée en construisant un modèle par homologie 3D de cette enzyme et par le docking de certains dérivés dans le site actif du CYP2J2. Enfin, une étude préliminaire des rôles biologiques possibles du CYP2J2 a été réalisée en étudiant les effets inhibiteur de ce P450. En conclusion, ce travail a permis de caractériser les premiers outils biochimiques d'étude des rôles biologiques du CYP2J2, de proposer une première topologie du site actif du CYP2J2, et d'affiner les rôles biologiques du CYP2J2.
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Dugrand-Judek, Audray. "Contribution à l’étude phytochimique et moléculaire de la synthèse des coumarines et furocoumarines chez diverses variétés d’agrumes du genre Citrus." Thesis, Université de Lorraine, 2015. http://www.theses.fr/2015LORR0238/document.
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Les coumarines et furocoumarines sont des phytoalexines synthétisées par certaines familles de plantes (ex : Rutacées dont font partie les agrumes), pour se défendre contre les bioagresseurs. Les furocoumarines peuvent être toxiques pour l’homme, lorsqu’elles sont combinées à certains médicaments : c’est l’effet pomelo. Aujourd’hui, la plupart des cytochromes P450 impliqués dans la synthèse des furocoumarines chez les Apiacées, ont déjà été caractérisés. En revanche, malgré l’importance économique des agrumes, nous en savons très peu sur la voie de biosynthèse des coumarines et furocoumarines chez ces plantes. Dans ce travail, nous avons créé, optimisé et validé une méthode d’analyse en chromatographie liquide à ultra haute performance couplée à un spectromètre de masse (UPLC-MS), pour identifier et quantifier 28 coumarines et furocoumarines dans la peau et la pulpe d’agrumes. Cette méthode nous a permis de chémotyper 62 variétés d’agrumes, distinguées par leur faible ou forte capacité de production de ces composés. En parallèle, un travail de bioinformatique sur des banques publiques d’ADN génomique d’agrumes, a permis d’identifier sept gènes présentant de fortes homologies de séquences avec ceux intervenant dans la synthèse des furocoumarines chez Pastinaca sativa (CYP71) et chez Arabidopsis thaliana (CYP82). Une analyse quantitative de leur niveau d’expression chez des agrumes, a montré que quatre d’entre eux étaient plus fortement exprimés chez les fruits fortement producteurs de coumarines et furocoumarines. Le clonage de ces gènes et leur expression hétérologue chez la levure, a révélé la fonction de CYP82D64 de pomelo et de Combava, qui hydroxyle la xanthotoxine pour donner la 5-hydroxy-xanthotoxine. La synthèse des coumarines et furocoumarines chez les agrumes, ainsi mieux appréhendée, nous a permis de proposer un schéma de sélection variétale visant à abaisser les taux de ces composés chez les Citrus. Nous avons aussi montré l’évolution convergente des CYP71 et CYP82 dans leur synthèse chez les Apiacées et les Rutacées respectivement. La découverte du premier cytochrome P450 de Citrus intervenant dans la production de ces composés, ouvre de nouvelles perspectives quant à l’élucidation de leur voie de biosynthèse chez les agrumesCoumarins and furanocoumarins are phytoalexines synthesized by some plant families (e.g. Rutaceæ that include citrus), to defend themselves against bioaggressors. Furanocoumarins can be toxic for humans, when combined with some drugs: this is the grapefruit juice effect. Nowadays, most of the cytochrome P450s involved in the furanocoumarin synthesis in Apiaceæ, have already been characterized. However, despite the economical importance of citrus, a little is known about the coumarins and furanocoumarins pathway in these plants. In this work, we created, optimized and validated an analytical method by ultra high performance liquid chromatography coupled with mass spectrometry (UPLC-MS), to identify and quantitate 28 coumarins and furanocoumarins in citrus peel and pulp. This method allowed us to chemotype 62 citrus varieties, distinguished by their low or high capacity to produce these compounds. In parallel, a bioinformatic work on public banks of genomic DNA from citrus, allowed to identify seven genes with high sequence homologies with those involved in the synthesis of furanocoumarins in Pastinaca sativa (CYP71) and in Arabidopsis thaliana (CYP82). A quantitative analysis of their expression level in citrus showed that four of them were more expressed in high coumarins and furanocoumarins producing fruits. The cloning of these genes and their heterologous expression in yeast, revealed the function of grapefruit and Combava CYP82D64, which catalyzes the hydroxylation of xanthotoxin in 5-hydroxy-xanthotoxin. The synthesis of coumarins and furanocoumarins in citrus, then better apprehended, allowed us to propose a breeding scheme aiming at decreasing the levels of these compounds in Citrus. We also showed the convergent evolution of CYP71 and CYP82 in their synthesis in Apiaceæ and in Rutaceæ respectively. The discovery of the first cytochrome P450 from Citrus involved in the production of these compounds, opens up new prospects for the elucidation of their biosynthetic pathway in citrus
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Krieger, Célia. "Identification moléculaire et caractérisation fonctionnelle d'une nouvelle sous-famille de cytochromes P450, CYP71AZ, impliquée dans la synthèse de furanocoumarines et coumarines chez Pastinaca sativa." Thesis, Université de Lorraine, 2014. http://www.theses.fr/2014LORR0185/document.
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Les furanocoumarines (FCs) sont des métabolites secondaires principalement synthétisés chez quatre familles botaniques et dérivent de la voie de biosynthèse des phénylpropanoïdes. Ces phytoalexines interviennent dans les processus de défense de la plante et présentent un fort potentiel thérapeutique. Des travaux réalisés dans les années 1960 sur des cultures cellulaires en parallèle de l’utilisation de précurseurs radiomarqués ont permis de démontrer que de nombreuses enzymes impliquées dans cette voie appartenaient à la famille des cytochromes P450 (P450s). Seules deux d’entre elles avaient pu être identifiées d’un point de vue moléculaire au début de ce travail de thèse. Afin de générer des informations concernant le génome de plantes productrices de FCs, nous avons fait séquencer les ARNm extraits de feuilles de Pastinaca sativa, de Ruta graveolens et de Cullen cinereum. L’analyse in silico de ces trois banques de données a permis d’identifier près de 800 fragments d’ADNc codants pour des P450s. Des travaux antérieurs réalisés au laboratoire et l’analyse comparative des transcriptomes de ces 3 plantes nous ont amenés à nous focaliser sur la sous-famille CYP71AZ au travers d’une étude fine de CYP71AZ3 et CYP71AZ4. La caractérisation fonctionnelle de ces enzymes a été réalisée dans un système d’expression hétérologue eucaryote : Saccharomyces cerevisiae. Les résultats obtenus ont permis de montrer que CYP71AZ4 avait une spécificité de substrat assez large puisqu’elle pouvait métaboliser au moins une FC et 4 coumarines. L’analyse et la comparaison des constantes cinétiques pour chacun de ces substrats indiquent néanmoins que le psoralène est le substrat préférentiel. La caractérisation fonctionnelle de CYP71AZ3 a mis en évidence que cette enzyme pouvait hydroxyler l’esculétine, une coumarine, mais ne jouait aucun rôle dans la synthèse de FCs. Ces travaux mettent en évidence la diversité fonctionnelle au sein d’une même sous-famille enzymatique et permettent d’émettre des hypothèses nouvelles quant à l’apparition de cette voie de biosynthèse chez les Apiacées d’une part, et chez les autres familles botaniques d’autre partFuranocoumarins (FCs) are secondary metabolites mainly synthetized in four botanical families deriving from the phenylpropanoid biosynthetic pathway. These phytoalexins are involved in plant defense mechanisms and present strong therapeutic potential. Early studies in the 1960s based on cell cultures and the use of radiolabeled precursors have shown that many enzymes involved in this pathway belong to the cytochrome P450 family (P450s). Only two of them had been identified from a molecular point of view at the beginning of this thesis. In order to generate information regarding the genome of plants producing FCs, we sequenced the mRNA extracted from leaves of Pastinaca sativa, Ruta graveolens, and Cullen cinereum. In silico analysis of these three libraries identified nearly 800 cDNA fragments encoding for P450s. Previous studies in the laboratory and comparative transcriptome analysis of these three plants have led us to focus on the subfamily CYP71AZ through a detailed study of CYP71AZ3 and CYP71AZ4. Functional characterization of these enzymes was performed in an eukaryote heterologous expression system: Saccharomyces cerevisiae. The results showed that CYP71AZ4 had a broad substrate specificity enough as it could metabolize one FC and 4 coumarins. The analysis and comparison of the kinetic constants for each of these substrates indicate, however, that the preferred substrate is psoralen. The functional characterization of CYP71AZ3 showed that this enzyme could hydroxylate esculetin, a coumarin, but played no role in the synthesis of FCs. This study highlights the functional diversity within a single enzyme subfamily and allows to issue new hypotheses about the emergence of this biosynthetic pathway in Apiaceae on one hand, and among other botanical families on the other hand
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Billard, Alexis. "Fenhexamid : mode d’action et résistance chez le complexe d’espèces Botrytis SPP., responsable de la pourriture grise de la vigne." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112002.
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La lutte chimique est la principale méthode utilisée pour contrôler les maladies causées par les champignons phytopathogènes. Dans certains cas, desphénomènes de résistance envers les fongicides se développent au sein despopulations, altérant parfois l’efficacité des molécules. La compréhension du moded’action des fongicides et des mécanismes de résistance sous-jacents participe à élaboreret à adapter des stratégies de management anti résistance ; et ainsi permettre depérenniser la durée de vie des molécules. Le fenhexamid est un fongicide récent (BayerCropScience, 2000), avec un mode d’action unique. Il est le seul fongicide commercialisébloquant l’étape de C4-déméthylation de la biosynthèse de l’ergostérol. Plusieurs typesde résistance (naturelle et acquises) ont été détectées dans les vignobles européens chez lecomplexe d’espèces Botrytis spp. responsable de la pourriture grise de la vigne. Lestravaux développés durant la thèse s’inscrivent dans l’objectif de la caractérisation dumode d’action et de l’élucidation des mécanismes de résistance. Le premier axe s’estattaché à la caractérisation fonctionnelle de deux gènes impliqués dans la C-4déméthylation de la biosynthèse de l’ergostérol : le gène erg27 codant la 3-céto réductase,cible du fenhexamid, et le gène erg28 codant une protéine qui interagirait en partie avecla 3-céto réductase. Concernant la résistance au fenhexamid, il a été démontré que, pargénétique inverse, les mutations détectées dans le gène erg27 de différents types d'isolatsrésistants issus du vignoble (phénotypes de résistance HydR3- et HydR3+) conféraient larésistance. Par ailleurs, une analyse de fitness du phénotype le plus préoccupant(phénotype HydR3+) a été réalisée en conditions contrôlées sur des souches isogéniquesartificielles afin d’apporter une réponse sur la persistance possible de ces souches auvignoble. Une méthode fine de quantification moléculaire de ces mêmes isolats aégalement été mise au point pour faciliter le suivi de leur évolution et de la persistancedes populations naturelles à l’échelle des vignobles. Cette nouvelle méthode, nomméeASPPAA PCR, exploite le polymorphisme nucléotidique du gène erg27, à l’origine de larésistance. Enfin, la résistance naturelle au fenhexamid de l’espèce apparentée à Botrytiscinerea, appelée Botrytis pseudocinerea a été élucidée. La résistance au fongicide de cetteespèce a été expliquée par la combinaison de modifications de cible (mécanismeminoritaire) et d’une dégradation du fongicide par un cytochrome P450 nomméCyp68.4 (mécanisme majeur). Il s’agit de la première identification et caractérisationgénétique d’un mécanisme de résistance à un fongicide conférée par un processus dedétoxification chez un champignon phytopathogèneChemical control is the main method used to control diseases caused byphytopathogenic fungi. In some cases, the resistance phenomena towardfungicides occur within fungal populations, which might alter practicalefficiency of molecules. Understanding modes of action of fungicides andunderlying resistance mechanisms participate to the development and adaptationof management strategies against resistance, and thus help to sustain the life ofmolecules. Fenhexamid is a recent fungicide (Bayer CropScience, 2000), with aparticular mode of action. It is the only fungicide marketed blocking the C4-demethylation step of ergosterol biosynthesis. Several types of resistance (naturaland acquired) were detected in European vineyards in the Botrytis spp speciescomplex, causing grey mold disease. This work focused on the characterization ofthe mode of action and the elucidation of resistance mechanisms. The first aspectinvestigated the functional characterization of two genes involved in the C4-demethylation of ergosterol biosynthesis. The erg27 gene potentially encoding the3-keto reductase which is the fenhexamid target and the erg28 gene encoding aprotein that interact in part with the 3-ketoreductase. Concerning fenhexamidresistance, we shown by reverse genetics that mutations detected in the erg27 genefrom different resistant isolates from the vineyards (phenotypes HydR3- andHydR3+) confer resistance. Furthermore, a fitness analysis under controlledconditions on the most worrying resistant phenotype (HydR3+) was performed onisogenic artificial strains in order to predict the possible persistence of these strainsin vineyards. A fine molecular method to quantify these isolates was developed tofacilitate the follow-up of evolution and persistence of resistant populations in thevineyard. This new method, named ASPPAA PCR is based on the nucleotidepolymorphism of the erg27 gene, responsible for fenhexamid resistance. Finally,the natural resistance to fenhexamid of the related species to Botrytis cinerea, B.pseudocinerea, was elucidated. Fungicide resistance of this species is explained bythe combination of target site modifications (minor mechanism) and fungicidedegradation mediated by a cytochrome P450 named Cyp68.4 (major mechanism).This is the first characterization of a genetic resistance mechanism to a fungicideconferred by detoxification in a phytopathogenic fungus
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Buron, Charlotte. "Développement de nouveaux catalyseurs d’oxydation bioinspirés : greffage de complexes de fer(II) non hémiques sur électrodes d’or ou dans la β-lactoglobuline." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA112148/document.
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Dans une thématique de plus en plus importante qui est celle du développement durable, il est aujourd’hui nécessaire d’adapter les réactions chimiques aux contraintes écologiques. Le développement de catalyseurs existe depuis le début de la chimie. Cependant, la compréhension des mécanismes mis en jeu lors des réactions chimiques est beaucoup plus récente, et ce, grâce à l’apparition de techniques d’analyse qui permettent de sonder les systèmes à différentes étapes des réactions. De nombreux catalyseurs moléculaires ont été développés, avec de très bons résultats au niveau des nombres de cycles catalytiques et de la sélectivité des réactions. Toutefois, ces catalyseurs sont souvent constitués d’un centre métallique de type iridium, ruthénium, palladium, rhodium ou platine, qui sont des métaux chers non biocompatibles. D’autre part, les oxydations sont des transformations chimiques très importantes. Des conditions de réactions dures, avec des oxydants stœchiométriques, souvent toxiques ou nocifs, sont généralement utilisées. Au contraire, des systèmes biologiques sont capables d’effectuer l’oxydation de molécules organiques en utilisant le dioxygène de l’air, en présence uniquement de protons, d’électrons, dans des conditions physiologiques. Quel grand défi pour les chimistes que d’arriver à développer des systèmes capables de mimer les systèmes biologiques, avec des catalyseurs composés de métaux biocompatibles tels que le cuivre, le manganèse et le fer. Le développement de ces catalyseurs biomimétiques demande une très bonne compréhension des systèmes biologiques, où les première et seconde sphères de coordination sont primordiales pour l'efficacité et la sélectivité des réactions. De nombreux complexes de fer(II) ont été développés comme catalyseurs dans notre équipe lors de thèses précédentes. L’interaction d’oxydants chimiques avec ces complexes a été étudiée, et une partie de mon projet a été de modifier les ligands pour augmenter la stabilité des catalyseurs, permettant l’augmentation de la sélectivité et les rendements de l’oxydation du cyclohexane et de l’anisole. Deux autres projets ont nécessité la fonctionnalisation d’un ligand utilisé communément au laboratoire pour son greffage covalent sur une électrode d’or ou dans une protéine. Afin de contrôler l’apport d’électrons au centre métallique pour réaliser l'activation réductrice du dioxygène, le complexe fonctionnalisé a été greffé sur des électrodes d’or. Le greffage sur électrode d’or a permis de mettre en avant la formation d’une monocouche homogène. Les premiers tests de réactivité de la SAM avec le dioxygène ont été également effectués. D’autre part, dans le but d’améliorer les rendements ainsi que la sélectivité des réactions en catalyse d’oxydation, un autre complexe fonctionnalisé a été greffé covalemment dans une protéine. Le greffage du complexe dans la β-lactoglobuline a permis de développer une nouvelle méthode de dosage du complexe de fer au sein de la protéine. Il a été possible de générer un intermédiaire réactionnel Feᴵᴵᴵ-peroxo, et les premiers tests en catalyse d’oxydation du thioanisole ont montré que la métalloenzyme artificielle permet d’observer une énantiosélectivité intéressante par rapport au complexe en solutionAccording to sustainable development it is necessary to adapt chemical reactions to ecologic constraints. Oxidation reactions are useful transformations. Nevertheless, reaction conditions used are frequently harsh, with oxidants used in stoichiometric amount (often harmful or toxic), and lead to products formation with low selectivity. Biological systems such as metalloenzymes, are able to perform small organic molecule oxidation following O₂ activation. These reactions are achieved in physiological conditions, and with a high selectivity. Deciphering the reaction mechanism of these biological catalysts has stimulated the development of synthetic analogues such as non heme iron(II) complexes bearing amine/pyridine ligands. Reaction of these Fe(II) precursors with H₂O₂ or a single oxygen atom leads to formation of Fe(III)-OOH, Fe(III)-(O₂) and Fe(IV)=O, identified as potent oxidizing species in biological systems such as cytochromes P450. In this work, ligands were functionalized to graft iron(II) complexes on gold surface or in the β-lactoglobuline protein in order to use O₂ as oxidant or to improve yields and selectivity, respectively. Complexes grafted on gold surface were characterized by cyclic voltammetry, AFM and XPS. It has been demonstrated that it is possible to exchange exogenous ligands of the iron complex grafted on gold electrode. Preliminar reactivity tests using this grafted complex and O₂ were performed. A new artificial metalloenzyme was synthesized by covalent grafting of a functionalized iron(II) complex on β-lactoglobuline. The system was characterized, and a new method of iron(II) titration in the protein was devised. Using hydrogen peroxide, an Fe(III)-(η²-O₂) intermediate was generated and indentified in the biohybrid system, and catalytic thioanisole oxidation was observed. Interestingly, the sulfoxide product formation was shown to be enantioselective under these conditions
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Skinner, Michael Stephen. "Olfactory cytochrome P450." Thesis, University of Warwick, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307349.
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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.
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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.
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Al-Anizy, Mohammed. "Studies on cytochrome P450 4X1." Thesis, University of Nottingham, 2005. http://eprints.nottingham.ac.uk/10404/.
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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.
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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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Marsh, Rachael. "Cytochrome P450 studies in Aspergillus fumigatus." Thesis, University of Sheffield, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364267.
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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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Papagiannidou, Eleni. "Cytochrome P450-mediated metabolism of melatonin." Thesis, University of Surrey, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422928.
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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.
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Busi, Florent. "Pharmacogénétique du cytochrome P450 humain CYP3A5." Paris 5, 2005. http://www.theses.fr/2005PA05P621.
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Smith, Gillian. "Xenobiotic regulation of cytochrome P450 gene expression." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/20797.
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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.
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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.
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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.
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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.
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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.
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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.
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Fraser, David John 1968. "Isolation and characterization of cytochrome P450 3A26." Diss., The University of Arizona, 1998. http://hdl.handle.net/10150/288850.
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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.
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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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Bertrand-Thiébault, Céline. "Cytochromes P450 et tonus vasculaire." Nancy 1, 2004. https://tel.archives-ouvertes.fr/tel-00120305.
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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.
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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.
Full textAbstract:
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.
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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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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.
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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.
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Hukkanen, J. (Janne). "Xenobiotic-metabolizing cytochrome P450 enzymes in human lung." Doctoral thesis, University of Oulu, 2000. http://urn.fi/urn:isbn:9514258649.
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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.
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Westlind, Johnsson Anna. "Pharmacogenetics of human cytochrome P450 3A (CYP3A) enzymes /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-688-x.
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Hu, Yin. "Genetic polymorphism and regulation of cytochrome P450 2E1 /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3690-0.
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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.
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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.
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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.
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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.
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Fan, Ming Qi. "Studies of the structure of cytochrome P450 4A1." Thesis, University of Nottingham, 2002. http://eprints.nottingham.ac.uk/10400/.
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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.
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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.
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Tan, Hoon Leong. "Selective inhibitors of the cytochrome p450 enzyme CYP1B1." Thesis, De Montfort University, 2006. http://hdl.handle.net/2086/4295.
Full textAbstract:
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.
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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.
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