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1
Urata, Kouji. "Hydrogen-driven hydrocarbon oxidation by cytochrome P450 enzymes." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:d5ec728a-2aa8-4040-92b0-56dad59e6dc4.
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P450BM3 (CYP102A1) from Bacillus megaterium is a 119 kDa, 1,046-residue polypeptide with the FAD/FMN reductase domain fused to the C-terminus of the haem domain and, as such it is catalytically self-sufficient; only NADPH and oxygen are required for monooxygenase activity. P450BM3 is a sub-terminal fatty acid hydroxylase, but generations of CYP102A1 engineering allowed them to be used in, e.g. drug metabolism and alkane oxidation. This thesis describes protein engineering of P450BM3 and altering reaction conditions to enhance C1 â C8 alkane oxidation activities, with the long-term goal of oxidising methane, and using the hydrogen-driven cofactor regeneration system to drive these reactions. Catalytic hydrocarbon oxidation under mild conditions is highly desirable in fuel synthesis and energy applications. Methane is a greenhouse gas and its effect can be minimised if methane is selectively oxidised to methanol, which can be used as a liquid fuel or feedstock. The R47L/Y51F, KT2, I401P and A330P mutants, which previously showed higher activities for a wide range of substrates, were used as templates to build a library of mutants. The R47L/Y51F/KT2/A330P mutant (RT2/AP) showed total turnover number (TTN) of 680 ± 10 under atmospheric pressure at ambient temperature for propane oxidation, and the TTN improved by 16-fold under 5 bar propane pressure (TTN is defined as the maximum number of moles of substrate converted per mole of P450BM3). TTN values of 14,250 ± 1,370 (KU4/AP) and 920 ± 50 (KU3/AP) were observed under 5 bar propane and 8 bar ethane, respectively, at ambient temperature. The effect of adding an inert perfluorocarboxylic acid (PFC), which resembles the structures of natural substrates and constrains small alkanes to bind closer to the haem, was investigated. The R47L/Y51F/N70S/M237L/A328V mutant (RL/YF/NMA) with PFC11 gave a TTN of 13,590 ± 30 under 5 bar propane at ambient temperature. Higher TTNs of 26,320 ± 1,010 for propane and 1,440 ± 70 for ethane oxidation were observed for the RL/YF/NMA mutant at 4 °C due to improved aqueous alkane solubility. Octane and propane oxidations were performed using a hydrogen-driven NADP+ recycling system without changing the selectivity of products, although the observed propane oxidation activity was 15% of the glucose/GDH cofactor recycling system.
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Larsen, Aaron. "Harnessing the power of P450 enzymes: a chemical auxiliary-based approach to predictable P450 oxidations at inactivated C-H bonds." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107745.
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Enantioselective hydroxylation of one specific methylene in the presence of many similar groups is debatably the most challenging chemical transformation. Although chemists have recently made progress towards the hydroxylation of inactivated C-H bonds, enzymes like P450s (CYPs) remain unsurpassed in specificity and scope. The substrate promiscuity of many P450s is desirable for synthetic applications; however, the inability to predict the products of these enzymatic reactions and the poor activity and stability of these enzymes is impeding advancement. In chapter 2 of this thesis, we evaluate several strategies to improve the activity and stability of CYP3A4. These strategies include the immobilization of CYP3A4 inside molecular hydrogels and silica in addition to the chemical modification of CYP3A4 using various anhydrides. Although none of the strategies we investigate here greatly enhance the catalytic utility of the enzyme, CYP3A4 is shown to be highly tolerant to functionalization at a large number of surface residues and to the presence of extremely high concentrations of silica during catalysis. Recognizing the potential for enzymes containing small, hydrophobic active sites to catalyze Diels-Alder reactions, chapter 3 describes the design and application of several assays to evaluate the Diels-Alderase activity of CYP2E1. Although the presence of CYP2E1 is not found to increase the rates of the reactions we investigate here, the results do demonstrate that there is an interaction between one or more of the substrates and the enzyme at either the active site or at another binding pocket. In chapter 4, we evaluate 4 auxiliaries for their ability to direct CYP3A4 oxidations. When linked to substrates, several of these auxiliaries are shown to direct CYP3A4 oxidations at specific C-H bonds. Although the auxiliaries we explore here are found to be limited in utility, several important lessons are learned which we apply to the design of a next generation auxiliary to be discussed in the following chapter. In chapter 5, we demonstrate the utility of theobromine as a chemical auxiliary to control the selectivity of CYP3A4 reactions. When linked to substrates, inexpensive, achiral theobromine directs the reaction to produce hydroxylation or epoxidation at the fourth carbon from the auxiliary with pro-R facial selectivity. This strategy provides a versatile yet controllable system for regio-, chemo- and stereo-selective oxidations at inactivated C-H bonds and establishes the utility of directing auxiliaries to mediate the activity of highly promiscuous enzymes. Recognizing the importance of product recovery, chapter 6 evaluates molecularly imprinted polymers for the selective purification of theobromine-containing molecules. When used for the solid-phase extraction, these materials allow for the near complete recovery of theobromine-containing products and starting materials from biocatalytic mixtures. This strategy represents an easily-tailored, effective, and reusable method of improving the recovered yield of theobromine-directed CYP3A4 oxidations.L'hydroxylation énantiosélective d'un méthylène spécifique en présence de nombreux autres groupes semblables est défendablement la transformation chimique la plus difficile. Bien que les chimistes aient récemment fait du progrès vers l'hydroxylation de liaisons C-H inactivées, il existe des enzymes comme les cytochromes P450 (CYP) qui demeurent inégalées par rapport à leur spécificité et leur portée. La promiscuité de substrat démontré par plusieurs P450 est souhaitable pour certaines applications de synthèse, mais une prévisibilité des produits difficiles, en plus de leur faible activité et stabilité empêchent l'avancement dans ce domaine. Dans le chapitre 2 de cette thèse, nous évaluons plusieurs stratégies pour améliorer l'activité et la stabilité de CYP3A4. Ces stratégies comprennent son immobilisation à l'intérieur d'hydrogels moléculaire et de silice, en plus de sa modification chimique avec une variété d'anhydrides. Bien qu'aucunes des stratégies étudiées ici n'aient grandement amélioré l'utilité catalytique de l'enzyme, elles démontrent quand-même que CYP3A4 est très tolérantes envers la fonctionnalisation d'un grand nombre de ses résidus de surface et à la présence de concentrations extrêmement élevées de silice pendant la catalyse. Reconnaissant le potentiel des enzymes possédant de petits sites actifs hydrophobes de catalyser des réactions Diels-Alder, chapitre 3 décrit la conception et l'application de plusieurs tests pour évaluer l'activité Diels-Alderase de CYP2E1. Bien que la présence de CYP2E1 n'a pas augmenter le taux des réactions étudiées ici, les résultats démontrent qu'il existe une interaction entre un ou plusieurs des substrats et l'enzyme, soit au site actif ou à un autre poche de liaison.Dans le chapitre 4, nous évaluons quatre auxiliaires pour leur capacité de diriger des oxydations par CYP3A4. Quand ils sont reliés à des substrats, nous avons trouvé que plusieurs de ces auxiliaires dirigent les oxydations par CYP3A4 à des liaisons C-H spécifiques. Bien que les auxiliaires explorés ici se trouvent à être limitées dans leur utilité, nous avons appris plusieurs leçons importantes que nous avons appliqué envers la conception d'une nouvelle génération d'auxiliaires à être discutés dans le chapitre suivant. Dans le chapitre 5, nous démontrons l'utilité de la théobromine en tant qu'auxiliaires chimique pour contrôler la sélectivité des réactions du CYP3A4. Quand il est relié à des substrats peu coûteux, la théobromine achiral dirige la réaction, donnant naissance à des produits hydroxylés ou époxydés au niveau du quatrième carbone à partir de l'auxiliaire avec une sélectivité faciale pro-R. Cette stratégie fournit un système versatile et contrôlable offrant des produits oxydés de façon regio-, chimio- et stéréo-sélective à des liaisons CH inactivé. Elle démontre aussi l'utilité des auxiliaires par rapport à leur habileté de contrôler l'activité des enzymes hautement promiscues. Reconnaissant l'importance de la récupération du produit, le chapitre 6 évalue des polymères à empreintes moléculaires pour la purification sélective de molécules contenant la théobromine. Quand ils sont utilisés pour l'extraction en phase solide, ces matériaux permettent la récupération quasi-complète de produits et de substrats contenant la théobromine à partir de mélanges biocatalytiques. Cette stratégie représente une méthode adaptable, efficace et réutilisable permettant d'améliorer le rendement récupéré des produits d'oxydations dirigée par la théobromine.
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Rodriguez, Patricia Fernandez. "Streamlined synthesis of taxol analogues." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:58d4a7f3-038e-4c4a-9aec-67267277670f.
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This thesis centres on the synthesis of taxol analogues via late-stage hydroxylation with P450 enzymes. To accomplish this, the taxane core, specifically taxa-4(5),11(12)-dien-2-one, was synthesised by classical synthetic methods, and subsequently oxidised using P450BM3 mutants. Chapter 1 introduces enzymatic catalysis, and the advantages and disadvantages of its application to organic synthesis. Additionally, an overview of taxol, including its discovery, mode of action, biosynthesis and large-scale production, and a summary of the previously reported approaches to the taxane core are described. Chapter 2 details the problems encountered and solutions implemented when reproducing Baran's route to taxa-4(5),11(12)-dien-2-one. Furthermore, approaches to some of its intermediates and an alternative route to taxa-4(5),11(12)-dien-2-one, which is based on Baran's, are discussed. Chapter 3 describes the development of a new, practical and short synthetic route to taxa-4(5),11(12)-dien-2-one which, ultimately, led to 1,3-di-epi-taxa-4(5),11(12)-dien-2-one. Additionally, the application of this route to the synthesis of a model compound and attempts to convert this racemic synthesis into an enantioselective route are reported. Finally, the enzymatic oxidation of taxa-4(5),11(12)-dien-2-one and related molecules using P450BM3 mutants is explored in Chapter 4. A preliminary study to determine the substrate enantioselectivity of the mutants is also described, along with the biological assays of the oxidised compounds produced during the study.
4
Celik, Haydar. "Enzyme-catalyzed Reductive Activation Of Anticancer Drugs Idarubicin And Mitomycin C." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/3/12609247/index.pdf.
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Idarubicin (IDA) and mitomycin C (MC) are clinically effective quinone-containing anticancer agents used in the treatment of several human cancers. Quinone-containing anticancer drugs have the potential to undergo bioreduction by oxidoreductases to reactive species, and thereby exert their cytotoxic effects. In the present study, we investigated, for the first time, the potential of IDA, in comparison to MC, to undergo reductive activation by NADPH-cytochrome P450 reductase (P450R), NADH-cytochrome b5 reductase (b5R) and P450R-cytochrome P4502B4 (CYP2B4) system by performing both in vitro plasmid DNA damage experiments and enzyme assays. In addition, we examined the potential protective effects of some antioxidants against DNA-damaging effects of IDA and MC resulting from their reductive activation. To achieve these goals, we obtained P450R from sheep lung, beef liver and PB-treated rabbit liver microsomes, b5R from beef liver microsomes and CYP2B4 from PB-treated rabbit liver microsomes in highly purified forms.
The plasmid DNA damage experiments demonstrated that P450R is capable of effectively reducing IDA to DNA-damaging species. The effective protections provided by antioxidant enzymes, SOD and catalase, as well as scavengers of hydroxyl radical, DMSO and thiourea, revealed that the mechanism of DNA damage by IDA involves the generation of ROS by redox cycling of IDA with P450R under aerobic conditions. The extent of DNA damages by both IDA and MC were found to increase with increasing concentrations of the drug or the enzyme as well as with increasing incubation time. IDA was found to have a greater ability to induce DNA damage at high drug concentrations than MC. The plasmid DNA experiments using b5R, on the other hand, showed that, unlike P450R, b5R was not able to reduce IDA to DNA-damaging reactive species. It was also found that in the presence of b5R and cofactor NADH, MC barely induced DNA strand breaks. All the purified P450Rs reduced IDA at about two-fold higher rate than that of MC as shown by the measurement of drug-induced cofactor consumption. This indicates that IDA may be a more potent cytotoxic drug than MC in terms of the generation of reactive metabolites. The results obtained from enzyme assays confirmed the finding obtained from plasmid DNA experiments that while MC is a very poor substrate for b5R, IDA is not a suitable substrate for this enzyme unlike P450R. The reconstitution experiments carried out under both aerobic and anaerobic conditions using various amounts of CYP2B4, P450R and lipid DLPC revealed that reconstituted CYP2B4 produced about 1.5-fold and 1.4-fold rate enhancements in IDA and MC reduction catalyzed by P450R alone, respectively. The present results also showed that among the tested dietary antioxidants, quercetin, rutin, naringenin, resveratrol and trolox, only quercetin was found to be highly potent in preventing DNA damage by IDA.
These results may have some practical implications concerning the potential use of P450R as therapeutic agent on their own in cancer treatment strategies. Selective targeting of tumor cells with purified P450R by newly developed delivery systems such as using polymers, liposomes or antibodies may produce greater reductive activation of bioreductive drugs in tumor cells. Consequently, this strategy has a high potential to increase the efficacy and selectivity of cancer chemotherapy.
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Leclaire, Jacques. "Oxydations catalysees par les cytochromes p-450 et les systemes metalloporphyriniques modeles : cas des alcenes monosubstitues et du leucotriene b::(4)." Paris 6, 1987. http://www.theses.fr/1987PA066473.
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La selectivite des oxydations catalysees par les monooxygenases a cytochrome p-450 tient compte de deux facteurs principaux: 1) le role de la chaine proteique bordant le site actif du cytochrome p450. 2) la chimioselectivite du complexe a oxygene actif implique dans la reaction. Oxydation du phenoxy-6 hexene 1 et du leucotriene b4 fait intervenir des familles de cytochromes p-450 tres differentes. L'utilisation de systemes metalloporphyriniques permet de faire, en partie, la difference entre les facteurs dus a la reactivite intriseque de l'entite a oxygene actif et ceux dus a l'environnement proteique. L'apoproteine est absente et la reconnaissance du substrat est reduite a son strict minimum puisqu'elle n'est plus excitee que par la metalloporphyrine. Mise au point de catalyseurs selectifs d'oxydation des olefine monosubstituees
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Adams, Delyth Ann. "Characterisation of human hepatic P450 enzymes." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266507.
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Schulz-Utermoehl, Timothy. "Identification and inhibition of hepatic p450 enzymes." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313273.
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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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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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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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Kelly, Paul. "Engineering cytochrome P450-reductase fusion enzymes for biocatalysis." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/engineering-cytochrome-p450reductase-fusion-enzymes-for-biocatalysis(c4b3aa48-1c73-4980-b480-e3a881267ee5).html.
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Cytochromes P450 (P450s) are a superfamily of heme-thiolate monooxygenases. They catalyse a wide variety of reactions on a vast number of substrates and are of particular interest for biocatalyst development due to their ability to oxidise non-activated C-H bonds. Fusion of a P450 to a suitable redox partner protein produces a catalytically self-sufficient enzyme and removes the need to produce electron transfer proteins separately. The well-studied bacterial protein P450cam (Pseudomonas putida) has been fused to the reductase (RhFRed) from the natural fusion protein P450-RhF (Rhodococcus sp.). The P450cam-RhFRed system catalyses the oxidation of camphor and several non-natural substrates and served as the basis for P450cam re-engineering in this current project, with the aim of expanding the substrate scope towards a more mammalian-like activity. The P450cam active site was partitioned into seven paired amino acids and each pair randomised in turn to generate seven sub-libraries of P450cam variants. These were screened for activity using a specially developed colony screen for detection of the blue pigment indigo. In total 94 new variants were identified and then pooled for secondary screening on a number of new substrates, identifying potentially novel activities within the ‘indigo positive’ population. In a separate ‘chimeragenesis’ approach substrate recognition sites (SRSs) within P450cam were targeted for exchange with equivalent portions from a number of human P450s. The B’ helix and F-G loop regions from CYPs 1A2, 2C8, 2D6 and 3A4 were grafted onto the P450cam structure and several of the B’ helix swaps were produced as soluble proteins. The P450cam-2C8-B’-RhFRed chimera gave a Soret peak at 420 nm in the Fe(II)-CO state although an additional substitution next to the proximal cysteine appeared to restore a P450-like state. SRS-exchange therefore offered some insight into structural modularity in P450s, providing a basis for further biocatalyst development.
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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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Ogilvie, Isla Marion. "Enzymes of mitochondrial #beta#-oxidation." Thesis, University of Newcastle Upon Tyne, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384937.
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O'Hanlon, Jack. "Evolved P450 mutants as general oxidation catalysts." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:a1bef635-0a21-4d22-b551-1f74574ac2f3.
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The thesis describes the use of a library of mutant P450BM3 variants for the biocatalytic oxidation of a range of structurally diverse substrates and the efforts toward the total synthesis of trigoxyphin K and L, employing biocatalytic C-H oxidation as a key step. Chapter 1 explores the P450BM3-catalysed oxidative cyclisation of α-amino (thio)acetamides for the synthesis of bicyclic imidazolidin-4-(thi)ones with P450BM3 variantcontrolled regio- and diastereoselectivity attainable in some cases. A method was developed for the Lewis acid-mediated ring-opening functionalisation of these compounds employing a variety of nucleophiles. Chapter 2 discusses the P450BM3-catalysed para-hydroxylation of three sulfonanilidederived herbicides and the expansion of this transformation to a number of substituted anilides. Regioselectivity of hydroxylation was possible for certain substrates, with selectivity possible for para-hydroxylation or benzylic hydroxylation. Chapter 3 details the successful approach to the core structures of the natural products trigoxyphin K and L and an investigation into the use of the library of mutant P450BM3 variants for late-stage oxidation of intermediates to enable synthesis of the natural products themselves. Finally, the P450BM3-mediated oxidation of a structurally diverse collection of small molecules and commercially available herbicides was examined with transformations including benzylic hydroxylation, acetal formation, S-oxidation, and nitro-group reduction.
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Hosseinpour, Fardin. "Cytochrome P450 Enzymes in the Metabolism of Vitamin D3." Doctoral thesis, Uppsala universitet, Institutionen för farmaceutisk biovetenskap, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-1791.
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A cytochrome P450 enzyme in pig kidney that catalyzes the hydroxylations of vitamin D3 and C27-sterols was cloned. DNA sequence analysis of the cDNA revealed that the enzyme belongs to the CYP27 family. The recombinant kidney CYP27A enzyme catalyzed the 25-hydroxylation of vitamin D3 and the 27-oxygenation of C27-sterols. It was shown that human embryonic kidney cells express CYP27A mRNA and are able to catalyze the same reactions. Microsomal vitamin D3 25-hydroxylase (CYP2D25), purified from pig liver, converted vitamin D3 into 25- hydroxyvitamin D3 in substrate concentrations which are within the physiological range. The enzyme also converted tolterodine, a substrate for CYP2D6, into its 5-hydroxymethyl metabolite. RT-PCR experiments revealed that CYP2D25 mRNA is expressed not only in liver and kidney but also in other organs. Experiments with human liver microsomes and recombinant human CYP2D6 indicate that the microsomal 25-hydroxylation of vitamin D3 in human liver is catalyzed by an enzyme different from CYP2D6. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Both wild-type and mutated CYP2D25 were expressed in the Saccharomyces cerevisiae W(R) strain. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained unaffected. These results indicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D3 metabolism. A cDNA homologous with the hepatic CYP2D25 was cloned from pig kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D3 and, in addition, lα-hydroxylation of 25-hydroxyvitamin D3. Immunohistochemistry experiments indicate that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. The results imply that CYP2D25 has a biological role in kidney. Results from experiments with inhibitors in primary cultures of porcine hepatocytes suggest that both CYP2D25 and CYP27A1 contribute to the total 25-hydroxylation in hepatocytes and are equally important in the bioactivation of vitamin D3. Phenobarbital treatment increased the CYP2D25 mRNA levels but did not affect the CYP27A1 mRNA levels. The rate of 25-hydroxylation by phenobarbital-treated hepatocytes was markedly reduced. These results show that primary cultures of porcine hepatocytes are suitable as a model to study the metabolism of vitamin D3 and the regulation of the CYP enzymes involved in the 25-hydroxylation o vitamin D3.
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Spellman, Ellen Frances. "Xenobiotic metabolism by cytochrome P450 enzymes in the brain." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247239.
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Hudson, Sean Andrew. "Fragment-based studies of mycobacterium tuberculosis cytochrome P450 enzymes." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607897.
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Jackson, Sandra. "Enzymes of mitochondrial fatty acid oxidation." Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283069.
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Talmann, Lea [Verfasser]. "Characterization of novel insect cytochrome P450-fusion enzymes / Lea Talmann." Gießen : Universitätsbibliothek, 2017. http://d-nb.info/1125851821/34.
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Rotzinger, Susan E. "Cytochrome P450 enzymes and the metabolism of trazodone and nefazodone." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0007/NQ34826.pdf.
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Coutts, Jane M. "Characterisation of mammalian cytochrome P450 enzymes by their metabolic activities." Thesis, Robert Gordon University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296198.
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Bathelt, Christine Michaela. "Reactivity and selectivity in cytochrome P450 enzymes and haem peroxidases." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422559.
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Żurek, Jolanta. "Modelling reactivity of threonyl-tRNA synthetase and cytochrome P450 enzymes." Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445804.
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Talmann, Lea [Verfasser]. "Characterization of novel insect cytochrome P450-fusion enzymes / Lea Talmann." Gießen : Universitätsbibliothek, 2021. http://d-nb.info/1239120710/34.
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Çelik, Ayhan. "Utilising haem enzymes in catalytic oxidation chemistry." Thesis, University of Leicester, 2001. http://hdl.handle.net/2381/30063.
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The catalytic oxidation of a range of substrates by recombinant pea cytosolic ascorbate peroxidase (rAPX), cytochrome P450 BM3 (P450 BM3) and poly-L-leucine has been examined and, for rAPX and P450 BM3, the results rationalised using computer-based molecular modelling. Transient and steady state kinetics, together with a range of chromatographic and spectroscopic techniques, have been used to establish the mechanism and the products of the H2O2-dependent oxidation of p-cresol by rAPX. The results are discussed in the more general context of APX-catalysed aromatic oxidations. The oxidation of a number of thioethers by rAPX and a site-directed variant of rAPX (W41A) has been examined. Steady state oxidation of the majority of sulphides studied, gave values for kcat that are approximately 10-fold to 100-fold higher for W41a than for rAPX. For rAPX, essentially racemic mixtures of R- and S-sulphoxides were obtained for all sulphides. The W14A variant shows substantial enhancements in enantioselectivity. Structure-based modelling techniques have provided a fully quantitative rationalisation of all the experimentally determined enantioselectivity and have indicated a role for Arg38 in the control of stereoselectivity. Cytochrome P450 BM3 is shown to be a versatile catalyst for the oxidation of a wide range of organic molecules. In this study we have shown that the enzyme is able to catalyse the regio- and stereo-selective oxidation of alkyl aryl sulphides to corresponding sulphoxides with varying degrees of enantioselectivity. The effects of both the nature of the alkyl group and the electronic properties of the aryl group on both rate an the stereoselectivity of the biotransformation have been explored. The results from this study have been compared to the theoretical prediction for the asymmetric induction expected for cytochrome P450 BM3 catalysed reactions. Poly-L-leucine proved to be a satisfactory catalyst for the oxidation of alkyl aryl sulphides, although this has been shown to occur with no significant asymmetric induction.
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Taylor, Samantha. "The modulation of cytochrome P450 activities by their membrane lipid environment." Thesis, University of Central Lancashire, 2001. http://clok.uclan.ac.uk/20846/.
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Cytochrome P450 (GYP) are a family of membrane-bound enzymes which form a component of the mixed function oxidase (MFO) system and are involved in the metabolism of many endogenous and exogenous compounds and, as such, play key roles in many physiological, pharmacological and toxicological processes. Due to the diversity of their substrates, it is essential to gain an understanding of how their activities can be modulated. The membrane lipid environment of CYP isoforms has proved to be an essential component for their optimal activities and consequently investigations into this aspect of modulation are fundamental to many areas of research. The work described in this thesis investigates 3 methods for the modulation of hepatic microsomal membrane lipids in order to relate 3 components of the lipid bilayer to the associated GYP isoform activities. The effect of incubating post-mitochondrial fractions with a range of phospholipase A2 (PLA2) concentrations at 37 °G on 4 CYP-catalysed reactions during microsomal fraction preparation was examined. The size of the membrane free fatty acid (FFA) pool was found to increase substantially following PLA2 treatment, and the main hydrolysis product was revealed to be arachidonate. Although the bulk fluidity of the membrane was unchanged at specific PLA2 concentrations, differential sensitivities of the GYP isoforms were observed at these concentrations. By assaying the activity of the NADPH-P450 reductase component of the MFO independently, in addition to using an oxygen surrogate to by-pass the reductase enzyme, it was concluded that it was specifically the GYP proteins that were susceptible to the membrane modulation which consequently lead to the observed decreases in the rates of reaction. The subsequent removal of the FFA pool with bovine serum albumin (BSA) was found not to restore the GYP isoform activities. It has been suggested that the inhibitory effect of FFAs may be due to conformational changes in the lipid environment of the isozymes. Also, hydrolysis of membrane phospholipids by PLA2 activity results in the formation of FFAs and Iysophospholipids, both of which can affect substrate partitioning within a membrane. Thus, the remaining lysophospholipids following BSA treatment may have contributed to the decreased activities of the GYP isoforms. A range of PLA2 concentrations at the lower incubation temperatures of 10 °G and 20 °C were used to retailor microsomal lipids in order to investigate the possibility that the GYP activities were associated with specific lipid domains. As with the 37°G incubation, the main hydrolysis product within the FFA pool was found to be arachidonate. Although arachidonate release was not significantly affected at any of the incubation temperatures, the activities of individual GYP isoforms were significantly decreased with increases in incubation temperature at specific PLA2 concentrations. Kinetic studies suggested that PLA 2 treatment of the microsomal membrane modulated CYP activity through the hydrolysis of associated lipid domains, and not by competitive inhibition by the endogenously released arachidonate. In an attempt to elucidate it microsomal membrane fatty acid saturation affected the activities of associated CYP isoforms, a preliminary study was conducted in which membrane lipid species were chemically modified by the use of catalytic hydrogenation. Membrane fractions were successfully hydrogenated, particularly long-chain fatty acids such asarachidonate. The activities of those CYP isoforms investigated were modulated to varying degrees upon hydrogenation of the membrane environment. It was concluded that the use of such catalysts should provide a suitable system for investigations fatty acid unsaturation and CYP activities. Finally, as phosphatidylcholine (PC) has been shown to be important for certain CYP isoform activities, a preliminary study was carried out to determine the effect of the biosynthetic pathway for PC synthesis on mouse hepatic MFO activity. Using microsomal fractions obtained from a transgenic strain in which one of the pathways for PC synthesis was absent, it was found that the PC fatty acid profile was changed compared to that of the wildype fractions. Additionally, the activities of certain CYP isoforms were affected by the biosynthetic origin of PC synthesis. This sensitivity of specific isozymes to the PC biosynthetic pathway may have also been sex-dependent. The work described in this thesis suggests several strategies which could be exploited further to refine and improve experimental studies of CYP isoforms. The results obtained support the concept that the membrane composition and environment of CYP isoforms differentially affects their activities. Due to the complexity of these remarkable biochemical systems, further studies are clearly required to define the membrane properties involved in the modulation of CYP activity.
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Norlin, Maria. "Cytochrome P450 Enzymes in the Metabolism of Cholesterol and Cholesterol Derivatives." Doctoral thesis, Uppsala University, Department of Pharmaceutical Biosciences, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-1086.
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Cholesterol is metabolized to a variety of important biological products in the body including bile acids and vitamin D. The present investigation is focused on enzymes that catalyze 7α-hydroxylation or 27-hydroxylation in the metabolism of cholesterol, oxysterols (side chain-hydroxylated derivatives of cholesterol) and vitamin D3. The enzymes studied belong to the cytochrome P450 enzyme families CYP7 and CYP27.
The study describes purification of a cytochrome P450 enzyme fraction active in 7α-hydroxylation of 25-hydroxycholesterol, 27-hydroxycholesterol, dehydroepiandrosterone and pregnenolone from pig liver microsomes. Peptide sequence analysis indicated that this enzyme fraction contains an enzyme belonging to the CYP7B subfamily. The purified enzyme was not active towards cholesterol or testosterone. Purification and inhibition experiments suggested that hepatic microsomal 7α -hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two enzymes, probably closely related.
The study shows that recombinantly expressed human and rat cholesterol 7α -hydroxylase (CYP7A) and partially purified pig liver cholesterol 7α -hydroxylase are active towards 20(S)-, 24-, 25- and 27-hydroxycholesterol. CYP7A was previously considered specific for cholesterol and cholestanol. The 7α -hydroxylation of 20(S)-, 25-, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. Cytochrome P450 of renal origin showed 7α -hydroxylase activity towards 25- and 27-hydroxycholesterol, dehydroepiaundrosterone and pregnenolone but not towards 20(S)-, 24-hydroxycholesterol or cholesterol. The results indicate a physiological role for CYP7A as an oxysterol 7α -hydroxylase, in addition to the previously known human oxysterol 7α -hydroxylase CYP7B.
The role of renal sterol 27-hydroxylase (CYP27A) in the bioactivation of vitamin D3 was studied with cytochrome P450 fractions purified from pig kidney mitochondria. Purification and inhibition experiments and experiments with a monoclonal antibody against CYP27A indicated that CYP27A plays a role in renal 25-hydroxyvitamin D3 l α -hydroxylation.
The expression of CYP7A, CYP7B and CYP27A during development was studied. The levels of CYP27A in livers of newborn and six months old pigs were similar whereas the levels of CYP7A increased. The expression of CYP7B varied depending on the tissue. The expression of CYP7B increased with age in the liver whereas the CYP7B levels in kidney showed a marked age-dependent decrease.
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Karlgren, Maria. "Novel extrahepatic P450 enzymes with emphasis on the tumor specific CYP2W1 /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-139-5/.
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Wepener, Ilse. "Pichia pastoris : a viable expression system for steroidogenic cytochrome P450 enzymes." Thesis, Stellenbosch : Stellenbosch University, 2005. http://hdl.handle.net/10019.1/50418.
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Thesis (MSc)--Stellenbosch University, 2005.ENGLISH ABSTRACT: This study describes: I. The cloning of the CVP 19 gene and construction of the intracellular expression vector pPIC3.5K-CYP19. II. The transformation of the yeast, Pichia pastoris with the constructed vector. III. The expression ofP450arom in Pichia pastoris. IV. The determination of enzyme activity and isolation of the protein from the Pichia pastoris cells. V. The expression of P450c 17 in Pichia pastoris. VI. The determination of kinetic constants for the conversion of progesterone to 170H-progesterone and 160H-progesterone by P450c17.
AFRIKAANSE OPSOMMING: Hierdie studie beskryf: I. Die klonering van die CVP 19 geen en die konstruksie van die intrasellulêre uitdrukkingsplasmied, pPIC3.5K-CYPI9. II. Die transformasie van die gis, Pichia pastoris, met die gekonstrueerde plasmied. III. Die uitdrukking van aromatase in Pichia pastoris. IV. Die bepaling van ensiemaktiwiteit en die isolering van die proteïen vanuit Pichia pastoris. V. Die uitdrukking van P450c17 in Pichia pastoris. VI. Die bepaling van kinetiese konstantes vir die omsetting van progesteroon na 170H-progesteroon en 160H-progesteroon deur P450c17.
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Perold, Helene. "The influence of Rooibos (Aspalathus linearis) on adrenal steroidogenic P450 enzymes." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/2381.
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Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009.This study: 1. Describes the preparation of unfermented and fermented rooibos methanol and aqueous extracts. 2. Investigates the influence of unfermented and fermented rooibos methanol and aqueous extracts on the binding of natural steroid substrates to ovine adrenal microsomal cytochrome P450 enzymes, demonstrating that the binding of natural steroids is inhibited in the presence of rooibos extracts. 3. Describes an assay demonstrating the inhibitory effect of rooibos extracts on the catalytic activity of cytochrome 17α-hydroxylase (CYP17) and cytochrome 21-hydroxylase (CYP21) in ovine adrenal microsomes. 4. Investigates the influence of unfermented and fermented rooibos methanol extracts on the catalytic activity of individual cytochrome P450 enzymes – CYP17 and baboon CYP21, that are expressed in COS1 cells. 5. Demonstrates that fractions of the unfermented rooibos methanol extract inhibits the binding of natural steroid substrate to microsomal cytochrome P450 enzymes as well as the catalytic activity of baboon CYP21 expressed in COS1 cells. 6. Investigates the inhibitory influence of individual rooibos flavonoids on the catalytic activity of baboon CYP21 expressed in COS1 cells.
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Faponle, Abayomi. "Biocatalytic studies of cytochrome P450 decarboxylase, related enzymes and biomimetic complexes." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/biocatalytic-studies-of-cytochrome-p450-decarboxylase-related-enzymes-and-biomimetic-complexes(1fcf5c03-c235-4985-892c-7aa02726308c).html.
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The biochemistry of cytochrome P450 is an interesting topic in biology and chemistry due to the importance of their functions to human health, but also has relevance to biotechnology. As such, understanding the catalytic mechanism of the P450s is crucial to assigning their applications. In this regard, it is often required to develop biomimetic models or synthetic analogues of their active sites for chemical investigation. In this thesis, I have investigated the mechanistic details of a novel cytochrome P450 Peroxygenase, OleTJE whose reaction with long chain fatty acids gives a mixture of alpha- and beta-hydroxy fatty acids and terminal alkenes. Our QM/MM calculation on its reaction mechanism reveals regioselective formation of these products and suggests how it can be bioengineered to alter the product distribution towards terminal alkene. We also studied the reactions of cytochrome P450 in drug metabolism and reveal the factors that determine the regioselectivity of substrate hydroxylation over desaturation in P450 isozymes. Spin-selective products formation was observed; the energy gaps between O-H and C-O bonds formed, and the pi-conjugation energy determines the extent of desaturation in addition to perturbations by environmental influences in the binding pocket as revealed by QM/MM study. The reaction of [(L52)FeIII(OOH)]2+ biomimetic model with aromatic compounds such as benzene and anisole as studied with DFT shows that hydroxylation occurs by direct C-O bond formation rather than an initial low-energy homolytic O-O bond cleavage which is slightly higher in energy. Moreover, the homolytic cleavage activates the oxidant toward reaction unlike in the heme where heterolytic cleavage occurs to form an active oxidant. This was followed by determining why phenol is a dominant product over ketone which is the primary product in reaction with aryl compounds. We also investigated the chemical and reactivity differences between nonheme iron(IV)-tosylimido and iron(IV)-oxo oxidants as biomimetic models of reactive intermediates in certain enzyme reactions. The iron(IV)-tosylimido complex has larger electron affinity and will react better with sulfides in an electrophilic addition than the iron(IV)-oxo which react faster in hydrogen atom transfer. These studies have employed computational analysis in most cases, and used to provide support for experimentally-obtained data where they exist; and have revealed fascinating bio(chemistry) of heme and nonheme iron-containing enzymes and oxidants with various substrates.
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Matheson, Johanne. "Heterologous expression and functional properties of plaice and human cypia-family enzymes." Thesis, University of Stirling, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244518.
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Sowden, Rebecca. "The oxidation of terpenoid hydrocarbons by monooxygenase enzymes." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288518.
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Hosseinpour, Fardin. "Cytochrome P450 Enzymes in the Metabolism of Vitamin D3." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5242-6/.
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Brown, Natasja. "Cape baboon Cytochrome P450 11β-hydroxylases : the characterization of two functional enzymes." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/19438.
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Dissertation (PhD)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: This study: 1. Describes the localization of CYP11B1 in the Cape baboon adrenal gland using Western blot analysis. CYP11B1 was localized to the adrenal cortex and medulla. 2. Describes the catalytic activity of CYP11B1 towards 11-deoxycorticosterone and corticosterone in adrenal cortical- and medullary tissue homogenates. Aldosterone formation in the adrenal medulla was identified using an atmospheric pressure chemical ionization-mass spectrometry method, which was developed in our department. 3. Compares the catalytic activity of three recombinant Cape baboon CYP11B1 cDNAs, expressed in COS-1 cells, towards 11-deoxycorticosterone and 11-deoxycortisol. 4. Describes the determination of the Michaelis-Menten constants and maximum reaction rates of 11-deoxycorticosterone and 11-deoxycortisol utilization by two functional recombinant Cape baboon CYP11B1 cDNAs, respectively. 11-Deoxycorticosterone metabolites were quantified using an enzyme immunoassay kit. 11-Deoxycortisol metabolites were quantified using a liquid chromatography-mass spectrometry method, which was developed in our department. 5. Describes the homology modeling of two isoforms of Cape baboon CYP11B1 using CYP102 and CYP2C5 as structural templates. The influence of three amino acid residue substitutions, located in the predicted D-E helix, on the catalytic activity of the two CYP11B1 isoforms was examined.
AFRIKAANSE OPSOMMING: Hierdie studie: 1. Beskryf die lokalisering van CYP11B1 in die bynier van die Kaapse bobbejaan deur gebruik te maak van die Western kladtegniek. CYP11B1 is gelokaliseer tot die adrenale korteks en medulla. 2. Beskryf die metabolisme van 11-deoksikortikosteroon en kortikosteroon in adrenale korteks- and medulla weefsel preparate, onderskeidelik. Die produksie van aldosteroon in die medulla is geïdentifiseer deur gebruik te maak van ‘n atmosferiese druk chemiese ionisasie-massa spektrometrie metode wat in ons departement ontwikkel is. 3. Vergelyk die katalitiese aktiwiteit van drie rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11- deoksikortisol metabolisme. 4. Beskryf die bepaling van die Michaelis-Menten konstantes en maksimum snelhede van twee funksionele rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11-deoksikortisol metabolisme. 11-Deoksikortikosteroon metaboliete is gekwantifiseer deur gebruik te maak van ‘n ensiem immunotoets. 11-Deoksikortisol metaboliete is gekwantifiseer deur middel van ‘n vloeistofchromatografie-massaspektrometrie metode, ontwikkel in ons departement. 5. Beskryf die modelering van drie-dimensionele strukture van twee funksionele Kaapse bobbejaan CYP11B1 isoensieme deur CYP102 en CYP2C5 as template te gebruik. Die effek van drie aminosuurresiduveranderinge in die voorspelde D-E heliks op die katalitiese aktiwiteit van die twee CYP11B1 isoforme is bepaal.
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Watts, Paul Michael. "P450 enzymes in the basal ganglia and their role in Parkinson's disease." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272165.
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Amadi, Cecilia Nwadiuto. "Biochemical and drug targeting studies of Mycobacterium tuberculosis cholesterol oxidase P450 enzymes." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/biochemical-and-drug-targeting-studies-of-mycobacterium-tuberculosis-cholesterol-oxidase-p450-enzymes(16cbca7a-b8b2-4ec4-bbd7-977785ed65b9).html.
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Mycobacterium tuberculosis (Mtb), a deadly pathogen, has scourged mankind for many centuries and has remained a major threat to global world health. Tuberculosis, the disease caused by this bacterium, is a major cause of death in developing nations and there is potential for its re-emergence in developed countries. An alarming rise in cases of multidrug-resistant and extremely-drug resistant tuberculosis (MDR-TB and XDR-TB) that do not respond to the customary first-line antibiotics necessitates the urgent need for development of new anti-TB drugs. Mtb becomes engulfed in human macrophages post infection of the host, but persists in the harsh environment of the human lungs by utilization of host cholesterol as a carbon source. The P450s CYP125A1, CYP142A1 and CYP124A1 are responsible for catalysing the side-chain degradation of cholesterol, which is critical for cholesterol to be used in the Mtb β-oxidation pathway for energy production. This PhD thesis focuses on understanding the structure/mechanism of the Mtb cholesterol 27-oxidases with the aim of facilitating the development of novel inhibitors of these P450s, which are crucial for Mtb to infect the host and to sustain infection. CYP142A1 and CYP124A1 were purified through three chromatographic steps with contaminating proteins successfully removed to give highly pure forms of these enzymes following the final purification step. Spectrophotometric titrations indicate that CYP142A1 and CYP124A1 bind tightly to cholesterol and cholestenone (and also to branched-chain methyl lipids for CYP124A1), highlighting their physiological roles in sterol and fatty acid metabolism, respectively. Binding analyses with a range of azole antibiotics revealed tight binding to bifonazole, clotrimazole, miconazole and econazole, and weak binding to fluconazole. Studies with compounds from a fragment screening library revealed weak binding to fragment hits for the cholesterol oxidases, but much tighter binding to these enzymes was found for ‘elaborated’ hits from a previous fragment screen on the Mtb cyclodipeptide oxidase CYP121A1, indicative of improved ligand potency achieved via ‘fragment merging’ strategies, and of structural similarities between these diverse Mtb P450s. Light scattering data indicate that CYP142A1 exists in dimeric form in solution, but becomes monomeric when treated with DTT; while CYP124A1 is completely monomeric. Crystal structures of CYP142A1 and CYP124A1 in complex with cholestenone, econazole and fragment library hits were determined. CYP142A1 crystal structures with econazole and fragment hits revealed heme coordination via the heterocyclic nitrogen in an azole group, and provide important data towards design of superior inhibitor drugs. The binding of cholestenone within the active site channels of CYP124A1 and CYP142A1 revealed an alignment favourable for C27 hydroxylation of the cholestenone side chain, which supports the physiological roles of CYP142A1 and CYP124A1 (as well as CYP125A1) in host cholesterol catabolism.
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Lightfoot, Tracy. "The structure and activity of cytochrome P450 2D6 (debrisoquine-4-hydroxylase)." Thesis, University of Sheffield, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310771.
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Maitland, Vivien. "Isozyme-specific induction of cytochrome P450 in rat hepatocyte cultures." Thesis, University of Aberdeen, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302485.
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The aim of this study was to investigate the induction of CYP1A by DMSO, to determine whether DMSO induced other P450 isozymes (CYP2B) and to compare the effects of DMSO and another differentiating agent, sodium butyrate. Induction of CYP1A-dependent ethoxyresorufin-O-deethylase (EROD) was observed in the presence of increasing concentrations of DMSO. All concentration investigated (1%, 1.5% and 2%) caused induction (2-3 fold), and enhanced BA-induction of EROD. Enhancement of BA-induction was greater with 1% and 1.5% DMSO (2.5-3 fold over BA alone) than with 2% (1.8-fold). DMSO alone did not increase CYP1A1 RNA levels. Hepatocytes treated with BA and DMSO together exhibited a 1.3-fold greater increase in RNA levels than with BA alone. Western blotting indicated that CYP1A1 protein was increased by inducers (BA, DMSO and isosafrole), but that CYP1A2 was not. This indicates that the CYP1A1 isozyme is responsible for EROD activity in these cultures, and that the CYP1A2-induction mechanism is lost in rat hepatocytes cultured under the conditions of these experiments. This observation was confirmed by the lack of CYP1A2-dependent phenacetin-O-deethylase (POD) activity in culture. The substituted benzimidazole omeprazole has been shown to induce CYP1A isozymes in human hepatocyte cultures. In this study omeprazole was not effective in inducing EROD activity in rat hepatocytes or in vivo in the rat. This confirms that rat hepatocytes are not a good model for CYP1A induction in man. DMSO appears to be isozyme specific, since CYP2B-dependent pentoxyresorufin-O-dealkylase (PROD) activity was not increased by DMSO, and phenobarbitone (PB) induction of PROD was enhanced only slightly by DMSO on day 3 of culture (4-fold over control; 1.5-fold over PB alone). Sodium butyrate and DMSO were both shown to induce differentiation of rat hepatocyte, with maintenance of low level of γ-glutamyl transferase activity, and maintenance of a more rounded morphology.
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Minn, Anne-Laure. "Caractérisation des enzymes et des transporteurs impliqués dans le métabolisme des xénobiotiques dans les tissus olfactifs." Dijon, 2005. http://www.theses.fr/2005DIJOPE01.
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Des enzymes du métabolisme des xénobiotiques sont présentes au niveau des tissus olfactifs, telles que les enzymes de phase I (cytochromes P450) mais également les enzymes de phase II (UDP-glucuronosyltransférases,. . . ). Elles participent à la détoxication des xénobiotiques mais également à l'éventuelle activation métabolique de ces molécules. Elles pourraient aussi jouer un rôle dans le processus physiologique de la perception des substances odorantes. La phase III est la phase d'excrétion des métabolites mais elle est méconnue au niveau de ces tissus. La présence des enzymes et protéines de phase I, II et III au sein des bulbes olfactifs et de la muqueuse olfactive démontre que ces tissus sont des lieux de métabolisme global non négligeables pour les molécules inhalées (polluants ou solvants). Cependant, cette voie reste intéressante du point de vue de l'administration de médicaments, notamment en vue d'un ciblage vers le cerveau, pour sa rapidité d'action et sa facilité d'utilisation.
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Deo, Anand K. "Hepatic microsomal bile acid biotransformation : identification of metabolites and cytochrome p450 enzymes involved." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/15222.
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Bile acids are end-products of cholesterol metabolism and essential for absorption of
dietary lipids in the body. Impaired bile flow leads to hepatic bile acid accumulation and liver damage. Hepatic microsomal oxidation offers a potential mechanism for efficient elimination of bile acids. The present study investigated the cytochrome P450 (P450)-mediated hepatic microsomal biotransformation profiles of lithocholic acid, cholic acid and chenodeoxycholic
acid using a liquid chromatography-mass spectrometry (LCIMS) based assay.
Incubation of lithocholic acid with rat hepatic microsomes resulted in the formation of a major 6β-hydroxylated metabolite, murideoxycholic acid, followed by isolithocholic acid and 3-ketocholanoic acid. Ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid were
identified as minor metabolites. Studies using P450-specific antibodies, chemical inducers, and rat recombinant enzymes showed that formation of murideoxycholic acid and 3-ketocholanoic acid were mediated by CYP3A2 and CYP2C 11. Similar metabolite profiles were obtained by incubation of lithocholic acid with mouse hepatic microsomes generating murideoxycholic acid
as the major metabolite. Studies using P450 inducers and chemical inhibitors suggested the involvement of murine CYP3A in murideoxycholic acid and 3-ketocholanoic acid formation, and CYP1A, CYP2B and CYP3A enzymes in ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid formation by mouse liver microsomes. Biotransformation of lithocholic
acid by human hepatic microsomes generated 3-ketocholanoic acid as the major metabolite, and hyodeoxycholic acid, ursodeoxycholic acid, 6-ketolithocholic acid and murideoxycholic acid, as minor metabolites. Studies with chemical inhibitors and human recombinant enzymes
demonstrated that CYP3A4 catalyzed the formation of all five metabolites.
The biotransformation of cholic acid and chenodeoxycholic acid by human hepatic
microsomes revealed the formation of a single cholic acid metabolite, 3-dehydrocholic acid. Chenodeoxycholic acid biotransformation generated 7α-hydroxy-3 -oxo-5β-cholan-24-oic acid as the major metabolite followed by у-muricholic acid, 7-ketolithocholic acid and cholic acid,
respectively. CYP3A4 was found to be the major enzyme involved in the biotransformation of cholic acid and chenodeoxycholic acid in human liver microsomes. A comparison of metabolite profiles demonstrated the dominant role of human CYP3A4 in the oxidation of bile acids at the C-3 position. In contrast, 6β-hydroxylation catalyzed by multiple P450 (CYP1A, CYP2B, CYP2C and CYP3A) enzymes was the preferred biotransformation pathway in rodent liver microsomes.
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Garcia, del Busto Elena. "Modulation of hepatic cytochrome P450 enzymes in two animal models of CNS inflammation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0029/MQ66566.pdf.
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Grimm, Scott Wayne. "Functional characterization of hepatic microsomal and heterologousely expressed rabbit cytochrome P450 2B enzymes." Diss., The University of Arizona, 1994. http://hdl.handle.net/10150/186952.
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The objective of the research described in this dissertation was to characterize the function of four closely related cytochrome P450 2B enzymes in the rabbit. Although these enzymes display greater than 97% amino acid sequence identity, their expression is highly variable between different organs and in different individuals. Transient and stable heterologous expression systems were used to study the distinct catalytic properties of each P450 2B enzyme. Cytochrome P450 2B5 was found to have a unique pattern of catalytic activities in comparison to the P450 2B4, 2B-B1, and 2B-Bx forms. The regio- and stereoselectivity of hydroxylation of androstenedione in hepatic microsomes depended upon whether the animal expressed cytochrome P450 2B5. Whereas the catalytic activities of P450 2B5 were characterized by high steroid hydroxylase activities, P450 2B4 had relatively low steroid hydroxylase activity and much higher activity towards the non-steroid substrates than 2B5. Androstenedione 15α-hydroxylation and benzyloxyresorufin O-debenzylation were identified as selective markers of the activity of P450 2B5 and 2B4, respectively. Phencyclidine selectively inactivated P450 2B4 in hepatic microsomes from phenobarbital-induced rabbits as well as the expressed enzyme. The basis for poor inactivation of P450 2B5 by PCP was determined to be the low maximal rate constant for this P450 2B form. N-Aralkylated 1-aminobenzotriazole derivatives were found to be potent inactivators of both P450 2B enzymes and to be much less selective than phencyclidine. These results demonstrate that one or more of the amino acid differences in P450 2B5 are critical to its substrate specificities and selective inactivation. Rabbits which express P450 2B5 have the potential to exhibit different hepatic biotransformation pathways in comparison with animals that lack this enzyme.
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Titus, Mark A. "Inhibition of human steroidogenic enzymes : steroid 5-alpha reductase and cytochrome P450 aromatase /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8178.
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Nishihara, Mitsuhiro. "Investigation of Drug Metabolism by Non-Cytochrome P450 Enzymes and Its Clinical Relevance." Kyoto University, 2014. http://hdl.handle.net/2433/189328.
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Gaudineau, Cédric. "Study of the activity of P450 2D6 in organic solvents and of the potential inhibition of P450 enzymes by food supplements." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82239.
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We investigated the ability of CYP2D6 to catalyze dextromethorphan O-demethylation in acetonitrile, chloroform, cyclohexane, and dichloromethane. Interestingly, in acetonitrile 20% activity was recovered.We next carried in vitro studies on the inhibitory effect of nicotinic acid and nicotinamide on human P450s involved in drug metabolism (CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4). At their therapeutic level, nicotinic acid inhibits CYP2D6 (Ki = 19 +/- 4 mM) whereas nicotinamide inhibits CYP2D6 (Ki = 3.8 +/- 0.3 mM), CYP2E1 (Ki = 13 +/- 8 mM) and CYP3A4 (Ki = 13 +/- 3 mM).
Finally, we have analyzed the different fractions (flavonoids and terpenoids) of a standardized Ginkgo biloba extract (EBG761) for their inhibition of the same human P450s as above. Our study shows that the flavonoidic fraction strongly inhibits these enzymes, especially CYP2C9 (Ki = 4.6 +/- 0.6 mM).
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Budde, Michael. "Biokatalyse mit Cytochrom P450 Monooxygenasen: zur selektiven Oxidation von Terpenen und Fettsäuren." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-32363.
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Maurer, Steffen Christian. "Oxidationsreaktionen mittels der Cytochrom P450-Monooxygenase CYP102A1 in Enzymreaktoren." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-28118.
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Souhaili-El, Amri Hajar. "Albendazole : métabolisme et régulation des enzymes hépatiques du métabolisme des xénobiotiques." Nancy 1, 1988. http://www.theses.fr/1988NAN10122.
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Ménard, Amélie. "P450 enzymes in biocatalysis: exploration of chemical auxiliaries, macromolecular crowding, bioconjugation and oriented-immobilization." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114213.
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Cytochrome P450 enzymes (CYPs or P450s) form a ubiquitous family of heme-dependent monooxygenases known mainly for their role in xenobiotic metabolism and their remarkable ability to regio- and stereoselectively oxidize inactivated C-H bonds, a feat that is difficult to achieve by chemical methods. Unfortunately, our ability to study and exploit these enzymes as in vitro biocatalysts has been limited by their low activity, low stability and poor product predictability. This thesis focuses on the study of human drug metabolizing P450 isoforms, namely CYP2E1, CYP3A4 and CYP2D6 because of their exceptional ability to accept a large variety of substrates. In Chapter 2, we demonstrate the utility of "type II ligands" as chemical auxiliaries for biocatalysis with human CYP2E1. We show that linking the chemical auxiliary nicotinate to a variety of short hydrocarbon substrates can promote their oxidation with predictable regioselectivity at the secondary aliphatic or alkenyl C-H bond furthest from the auxiliary. The origin of this selectivity was rationalized through docking studies of our auxiliary-substrate compounds with reported X-ray crystals structures of CYP2E1. These results not only confirm the general utility of the chemical auxiliary approach pioneered by our lab to direct the predictable oxidation of inactivated C-H bonds by P450 enzymes, but also provide a system with complementary regioselectivity.A short study of the effects of macromolecular crowding on the activity of human CYP3A4 and CYP2D6 is presented in Chapter 3. We found that certain crowding agents were not detrimental to enzyme activity while others had a negative effect. Moreover, certain conditions (initially tested as controls) that improved enzymatic activity were uncovered. In Chapter 4, the non-covalent oriented-immobilization of CYP3A4 via its C-terminal histidine-tag is described. We show that immobilization on Ni-NTA agarose resin via this strategy has no detrimental effect on enzyme activity or stability. The lyoprotectant properties of Ni-NTA were also investigated. In Chapter 5, we designed and characterized a mutant of CYP3A4 that retains its enzymatic activity upon modification with a variety of fluorescent maleimide dyes via a single cysteine residue on its surface, namely C64. We also show that the activity of this mutant is preserved upon immobilization onto solid supports via this same cysteine residue. Finally, results of a preliminary feasibility study towards applying this immobilization strategy to eventual single-molecule fluorescence microscopy studies are presented.Les enzymes cytochrome P450 (CYPs ou P450) forment une famille omniprésente de mono-oxygénases possédant un noyau hème au site-actif. Ces enzymes sont surtout connues pour leur rôle dans le métabolisme de produits pharmaceutiques et pour leur capacité remarquable à oxyder les liens C-H non-activés de façon régio- et stéréosélective. Malheureusement, notre capacité d'étudier et d'utiliser ces enzymes comme biocatalyseurs in vitro est limitée par leur faible activité, instabilité et une incapacité des connaissances actuelles à prédire leurs produits.Dans le chapitre 2, nous démontrons l'utilité des ligands de type II comme auxiliaires chimiques pour la biocatalyse avec CYP2E1 humaine. Nous démontrons que l'auxiliaire chimique nicotinate, lorsque lié à une variété d'hydrocarbures courts, peux promouvoir leur oxydation avec une régiosélectivité prévisible pour le lien C-H secondaire aliphatique ou alcényle le plus éloigné de l'auxiliaire. L'origine de cette sélectivité a été rationalisée à l'aide de «docking» moléculaire de nos composés auxiliaire-substrats à l'intérieure de structures cristallines de CYP2E1 publiées par d'autres chercheurs. L'utilité d'auxiliaires chimiques pour contrôler la régiosélectivité des enzymes P450 avait déjà été démontrée par notre groupe de recherche. Les résultats présentés dans ce chapitre offrent non seulement une confirmation du potentiel de cette stratégie, mais aussi un système complémentaire pour l'oxydation prévisible de liens C-H non-activés par les enzymes P450. Ces résultats confirment également la généralité de l'approche mis au point dans notre laboratoire qui décrit l'utilisation d'une auxiliaire chimique pour diriger l'oxydation prévisible de liens C-H non-activés par les enzymes P450.Une étude des effets de l'encombrement macromoléculaire sur l'activité enzymatique des CYP3A4 et CYP2D6 humaines est présentée dans le chapitre 3. Nous avons trouvé que leur activité demeure inchangée par la présence de certains agents encombrants alors que d'autres ont un effet négatif. De plus, certaines conditions (testées initialement comme contrôle) qui améliorent l'activité enzymatique ont été découvertes.Dans le chapitre 4, l'immobilisation orienté non-covalente de CYP3A4 par son étiquette de type his-tag C-terminale est décrite. Nous démontrons que son immobilisation sur une résine Ni-NTA à base d'agarose via cette stratégie n'a aucun effet négatif sur l'activité ou la stabilité de l'enzyme. Les propriétés lyoprotectrices de cette résine ont aussi été investiguées.Dans le chapitre 5, nous concevons et caractérisons un mutant actif de CYP3A4 lors de modifications avec une variété de maléimides fluorescentes à l'endroit d'un unique résidu cystéinique à sa surface, soit le C64. Nous démontrons aussi que ce mutant préserve son activité lorsqu'immobilisé sur des supports solides par ce même résidu cystéinique. Finalement, les résultats d'études préliminaires sont présentés qui envisagent l'application de cette stratégie d'immobilisation envers des études éventuelles de spectroscopie de fluorescence à la résolution d'une seule molécule.