Résumé : Le métabolisme monocarboné ou métabolisme 1-C désigne l’ensemble des voies métaboliques permettant la synthèse et / ou le recyclage de molécules donneur de groupement monocarboné au cours des réactions de méthylation. L’objectif de ce travail était d’évaluer l’implication des métabolismes de la choline, de la phosphatidylcholine (PC) et de la bétaïne dans la physiopathologie des désordres impliquant le métabolisme 1-C en période prénatale et postnatale. Nous avons montré une augmentation progressive de l’expression de la majorité des gènes impliqués dans le métabolisme 1-C au cours de l’ontogénèse hépatique murine, tandis que les leur expression était plus faible avec des profils plus variable au niveau cérébral. Chez l’homme, les valeurs normales des concentrations des intermédiaires du métabolisme 1-C dans le liquide amniotique (LA) en fonction du terme gestationnel ont été déterminées pour tous les paramètres et les concentrations de S-adénosyl-homocystéine et de méthionine étaient augmentées dans les LA du groupe affecté par des défauts de fermeture du tube neural (DFTN) suggérant que certains cas de DFTN pourraient être associés à des déséquilibres du métabolisme 1-C. En post natal, nous avons montré à la fois chez l’homme et l’animal, que les hyperhomocystéinémie d’origine nutritionnelles ou génétiques induisaient une déplétion en bétaïne, épargnant uniquement le rein où elle est un osmolyte majeur. Dans un modèle murin de déficit en cystathionine–beta synthase induisant une hyperhomocystéinémie, une technique de lipidomique ciblée a montré au niveau hépatique des modifications qualitatives des phospholipides (PLs) avec une diminution des PC contenant des acides gras insaturés et des phosphatidyléthanolmines contenant de l’acide arachidonique. Ces modifications des PLs pourraient jouer un rôle dans la constitution de la stéatose hépatique observée dans l’histoire naturelle de cette maladie. En conclusion, ce travail a permis de montrer que la choline, la bétaïne et les PC sont des acteurs indissociables du métabolisme 1-C qui pourraient être impliqués dans la physiopathologie des DFTN et dans les hyperhomocystéinémies. Ils pourraient également être impliqués dans la physiopathologie des stéatoses hépatiques non alcooliques ou des déficits cognitifs, dans lesquels des désordres du métabolisme 1-C ont été observés. / Abstract: One carbon metabolism or 1C metabolism includes all metabolic pathways for the synthesis and / or recycling of molecules involved in methylation reactions. The objective of this study was to evaluate the involvement of choline, phosphatidylcholine (PC) and betaine metabolisms in the pathophysiology of diseases with impaired 1-C metabolism in prenatal and postnatal period. We showed a progressive increase of the expression of the majority of genes involved in 1-C metabolism during the mouse liver ontogeny while their gene expression was at lower levels and with more variable patterns during brain ontogeny. In humans, amniotic fluid concentrations of all intermediates of 1-C metabolism according to gestational term were determined and we observed increased concentrations of S-adenosyl-homocysteine and methionine in pregnancies affected by neural tube defects (NTD) suggesting that some NTDs cases could be associated with an imbalance in 1-C metabolism. In the postnatal period we showed that both in animal and humans and both in nutritional and genetic hyperhomocysteinemia, that betaine pools were decreased, only sparing the kidney betaine concentrations, where betaine acts as an essential osmolyte. In a mouse model of cystathionine-beta synthase deficiency inducing hyperhomocysteinemia, a technic of targeted lipidomic revealed qualitative changes in the liver phospholipids composition, in particular a decrease of PC containing unsaturated fatty acids and of phosphatidylethanolamine containing arachidonic acid and an increase of phosphatidylethanolamine containing docosohaexaenoic acid. This phospholipids remodelage may participate in the development of the steatosis observed in the natural history of this disease. In conclusion, this study has shown that choline, betaine and phosphatidylcholine are essential actors of 1-C metabolism that could be involved in the pathogenesis of NTD and hyperhomocystéinemia. They could also be involved in the pathophgysiology of non alcoholic fatty liver disease or cognitive decline, in which disorders of 1-C metabolism were observed.

Dans cette thèse, j'ai essayé d'identifier le mode d'action de composés biscationiques récemment synthétisés et leurs interactions avec les parasites résistants à la chloroquine. Les activités de divers représentants des composés ammonium bisquaternaire, des amidines alkyl (provenant du groupe de professeur Vial, Montpellier, France), des amidines bisbenzyl (provenant du groupe Chimie de l'Université de Liverpool, Royaume-Uni) ont été comparés à la chloroquine et la pentamidine. Leurs potentiels de résistance croisée avec la chloroquine ont également été étudiés. Dans ce but, deux lignées cellulaires modifiées génétiquement par échange allélique, C3Dd2 et C2GCO3 furent utilisées.Parmi les amidines bisbenzyl, une série de composés appartenant aux guanidines, thiazoles et triazoles ont été criblés pour leur activité contre des souches résistantes et sensibles à la chloroquine chez Plasmodium falciparum. Une hypersensibilité significative est observée pour les amidines bisbenzyl parmi les isolats affichant un PfCRT mutant. Aucune différence n'est observée pour les composés provenant du groupe Vial. Pour comprendre le mode d'action et le rôle de PfCRT, j'ai réalisé des expériences de fixation compétitives (competitive binding') et de cristallisation d'hème. Tous les composés ont montré à différents degrés des interactions avec l'hème, cependant il fut observé que leur activité ne corrélait pas avec l'inhibition de la cristallisation d'hème. Une des raisons possibles à cela est que les différences structurales peuvent jouer un rôle important dans le transport du composé. De plus, j'ai étudié l'effet du pH sur l'activité des composés en utilisant les lignées cellulaires modifiées génétiquement par échange allélique afin d'observer l'effet du gradient de proton sur le transport de la chloroquine et de la pentamidine. Des différences significatives de l'activité de la chloroquine furent observées chez les deux souches. Malgré les valeurs de pKa élevées pour la pentamidine, il y avait une différence significative dans la sensibilité pour ce composé chez les souches quand le pH a changé.Car les diamidines requièrent des transporteurs pour traverser les barrières membranaires et qu'un possible transporteur de choline a été caractérisé chez Plasmodium falciparum, j'ai également réalisé des études initiales sur la caractérisation moléculaire de ce transporteur. Un gène qui encode une protéine chez P. falciparum avec une similarité significative aux eucaryotes supérieurs fut identifié en utilisant des analyses bioinformatiques et fut employé dans une transformation et des études analyses fonctionnelles.En conclusion, ce travail suggère qu'il est possible d'utiliser de nouveaux amidines bisbenzyl pour cibler spécifiquement les souches résistantes à la quinoléine chez Plasmodium falciparum, arborant des allèles de PfCRT mutantes. En adhérant à cette hypothèse et sachant que les deux classes de composés fixent la même cible non parasitaire (soit l'hème), il serait possible de créer rationnellement une combinaison de composés quinoléine / diamidine. Ainsi, les souches résistantes à un des deux composés seraient plus sensibles à l'autre partenaire, retardant ainsi l'apparition de résistance. / In this thesis I have attempted to subject the issues of mode of action of recently synthesized bis cationic compounds and their possible interactions with chloroquine resistance. Antimalarial activities of representatives of various bis quarternary ammonium compounds, alkyl amidines (received from Dr.Vial group, Montpellier) and of bisbenzyl amidines (received from Chemistry group, Liverpool) activity have been investigated with chloroquine and pentamidine and looked for cross resistance with chloroquine. For this purpose two genetically modified allelically exchanged cell lines C3Dd2 and C2GCO3 modified on the chloroquine resistance-related PfCRT (P.falciparum chloroquine Resistance Transporter) gene were used. Among the benzyl amidines, a significant hypersensitivity tobis benzyl amidines was observed among the isolates bearing the mutant PfCRT. No such difference is observed for the bisalkyl amidines. To understand the mode of action and role of PfCRT, competitive binding assay to heme (which may mediate the well-known cellular accumulation of the compounds) and effect on heme crystallization assays (which is involved in the toxic effect against the intracellular parasite) were performed. All these compounds were shown to interact with heme in various degrees. Their activity was observed not to be correlating with heme crystallization inhibition. This is likely due to the structural differences between the compound which discriminate the compounds in the transport of the compound to the parasite and their mechanism of antimalarial activities. In addition I have studied the effect of pH on the pharmacological activity of the drugs using allelically exchanged genetically modified cell lines (for PfCRT) to characterize the importance of proton gradient on the transport of chloroquine and pentamidine to the intracellular parasite. Significant difference (reduced antimalarial activity with increased pH) in the activity of chloroquine was observed for both the strains. Despite of the high pKa values for pentamidine, there was significant difference in the sensitivity of the strains to this compound, when the pH is changed. As both the diamidines and choline analogs require transporters to cross the membrane barriers and enter the parasite where they accumulate I have also performed initial studies on the molecular characterization of a potential carrier in P.falciparum. Using basic bioinformatic tools, a gene encoding a P.falciparum protein with significant similarity to higher eukaryotes choline transporter was identified and preliminary work for its functional analysis was performed. In conclusion, this work establishes substantial differences between the various classes of bis-cationic compounds essentially (based on benzamidine and choline-analogs alkylkamidine series) concerning their interaction with the infected erythrocyte and their antimalarial activity. The series are diffentallly affected by the PfCRT mutation and the chloroquine resistance. Results suggest that it may be possible to use novel bisbenzyl amidines to specifically target quinoline resistant Plasmodium falciparum malaria, harbouring mutant pfcrt alleles. Taking this idea further and since both classes of compound target the same non-parasite target (heme), it may even be possible to rationally design a quinoline / diamidine drug combination, in which isolates resistant to one partner drug become more sensitive to the other partner, thus delaying the onset of resistance.

No description available.

D-Arginine dehydrogenase (DADH) catalyzes the oxidation of D-arginine to imino arginine using FAD as the cofactor. The enzyme is part of a recently discovered two-enzyme complex from Pseudomonas aeruginosa involved in arginine utilization. Function of the enzyme within the organism is unknown. Work on this enzyme has been undertaken to understand the structure as well as its reaction mechanism so as to eventually assign a function to the enzyme within the physiological context. In the reductive half-reaction 2 e- and 1 H+ are transferred from the amino acid substrate to FAD cofactor. In the oxidative half-reaction the reducing equivalents from the FAD cofactor are passed to an electron acceptor that is yet to be discovered. The enzyme has been established to have no reactivity with O2. Choline oxidase (CHO) from Arthrobacter globiformis is a well characterized member of Glucose-Methanol-Choline Superfamily that reacts with molecular O2. It catalyzes the oxidation of choline to glycine betaine mediated by betaine aldehyde intermediate using FAD as the cofactor and O2 as the oxidant to regenerate oxidized FAD for further reaction. Glycine betaine, the product of the reaction is an important osmolyte that regulates nutrients for plants under stressful conditions. Therefore it is of commercial interest to genetically engineer crops that do not typically possess competent pathways for glycine betaine synthesis. In this dissertation molecular details concerning the reductive half-eaction of DADH and oxidative half-reaction of CHO have been studied using a combination of steady state kinetics, rapid kinetics, pH, multiple substrates, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects or computational approaches. In DADH, the oxidation of amino acid substrate by FAD has been shown to most likely proceed via hydride transfer mechanism in the reductive half-reaction with Glu87, Tyr53, Tyr249 and His48 emerging as key players in substrate binding, catalysis or for up keeping the integrity of the FAD cofactor. In CHO, the oxidative half-reaction proceeds without stabilization of any reaction intermediates with H atom from reduced FAD and H+ from solvent or solvent exchangeable site occurring in the same kinetic step.

: Choline is being used in all mammalian cells as a precursor for synthesis of a major phospholipide phosphatidylcholine and as a donor of acetyl residues. Cholinergic neurons in addition require choline to synthesize neuromediator acetylcholine. The ability of cells to create choline via de novo synthesis is limited and therefore they need to transport choline from extracellular space. Limited availability of choline in brain leads specifically to diminished function of cholinergic neurons and in general to impaired reparation of biological membranes. Dysfunctions of cholinergic signaling in brain is characteristic for Alzheimer's disease. Aim of this work was to investigate whether gene and protein expression of high- affinity cholinergic transporters is altered in 5-6 months old APPswe/PS1dE9 mouse model of Alzheimer's disease. Expression of specific high-affinity cholinergic transporter CHT1 (responsible for transport of choline to be used for acetylcholine synthesis) and putative high-afinity choline transporter CTL1 (generally present in all cells and related to high affinity choline transport for phospholipide synthesis) in cerebral cortex was measured. Compared to non-trangenic littermates, no changes in the expression of both genes were detected at either mRNA (quantitative PCR) or protein...

Le métabolisme des monocarbones relie le métabolisme cellulaire à la machinerie épigénétique à travers une molécule commune, la S-adénosylméthionine (SAM). Les changements dans le potentiel de méthylation cellulaire (ratio SAM/SAH) sont impliqués dans plusieurs maladies, notamment l'hépatocarcinome et les défauts de fermeture du tube neural (NTD). La perturbation du ratio SAM/SAH peut être liée à des déficits enzymatiques ou à une exposition à un facteur environnemental. La reméthylation de l'homocystéine (HCY) en méthionine est catalysée par la méthionine synthase (MTR) ou la bétaïne homocystéine méthyltransférase (BHMT) dans le foie. En comparant le tissu tumoral au tissu sain environnant dans le foie, les transcrits de BHMT étaient fortement diminués dans les échantillons mais pas les transcrits de MTR. La protéine BHMT n'a pas été détectée dans les cellules HepG2 et dans 5 des 6 échantillons tumoraux analysés. L'absence d'expression de BHMT était due à un variant génétique conduisant un codon stop prématuré. Une déficience pré-natale en donneurs de méthyle aggrave la susceptibilité face à certaines maladies. La fumonisine B1 (FB1) est une mycotoxine qui contamine les céréales et a été identifiée comme étant un facteur de risque de survenue de tumeur et des NTD. Nous avons étudié les récepteurs des folates et 4 marques de l'hétérochromatine dans le foie des foetus de rat issus de mères exposées à un régime déficient en donneurs de méthyle et/ou à la FB1 à une dose deux fois supérieure à la dose journalière admissible. Le régime carencé et la FB1 diminuent le ratio SAM/SAH. La FB1 inverse le mécanisme d'adaptation consistant en une régulation positive de l'expression des récepteurs des folates lors d'une carence seule. Le régime carencé diminue H4K20me3 mais une combinaison carence/FB1 diminue encore d'avantage H4K20me3 et augmente H3K9me3. Cette augmentation de H3K9me3 peut être vu comme un mécanisme de défense incitant la cellule à résister à la désorganisation de l'hétérochromatine. H3R2me2 et H4K16ac varient également selon ce mécanisme. Cette étude est pertinente car elle suggère que de faibles doses de FB1 interagissent avec la carence en donneurs de méthyle pour perturber le profil épigénétique / Folate-mediated 1-carbon metabolism is a conduit that links cellular metabolism to the epigenetic machinery through the common molecule, AdoMet. There is strong evidence that changes in the cellular methylation potential (AdoMet/AdoHcy ratio) is involved in several types of disease notably tumor proliferation like hepatocarcinoma and developmental disease like neural tube defects. Perturbation of AdoMet/AdoHcy ratio may be related to a cellular cause like enzyme defect or to exposure to an environmental factor. The remethylation of homocysteine to methionine is catalyzed either by methionine synthase (MTR) or by betaine-homocysteine methyltrnasferase (BHMT) in the liver. By comparing tumor tissue to surrounding healthy tissue in the liver we have found that BHMT transcripts, but not MTR, are strongly decreased in tumor samples. Consistently, BHMT protein was not detected in HepG2 cells and in 5/6 tumors investigated. Abolition of BHMT expression was due to a genetic variant producing a premature termination codon. Prenatal methyl deficient diet (MDD) enhances susceptibility to disease. Fumonisin FB1 is a corn contaminating mycotoxin identified as a risk factor for tumor occurrence and neural tube defects. We have investigated folate receptors and 4 heterochromatin markers in rat foetuses liver derived from dams exposed to MDD and/or FB1 administered at a dose twice higher than the Provisional Maximum Tolerable Daily Intake. We found that MDD and even FB1 by itself decrease the AdoMet/AdoHcy ratio. FB1 reverses the adaptation mechanism consisting in upregulating folate receptors in case of folate depletion. MDD decreased H4K20me3 but combined MDD/FB1 decreased H4K20me3 even more and increased H3K9me3. The elevated H3K9me3 can be viewed as a defence mechanism inciting the cell to resist heterochromatin disorganisation. H3R2me2 and H4K16Ac varied according to this mechanism. This study is relevant because it suggests that low doses of FB1 interact with methyl depletion to disrupt the epigenetic landscape

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-06-07T16:23:22Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Diego Andres Velasco Acosta.pdf: 1957910 bytes, checksum: 57eabc538f89e509cac0a69175bd4efb (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-08T13:04:26Z (GMT) No. of bitstreams: 2 Diego Andres Velasco Acosta.pdf: 1957910 bytes, checksum: 57eabc538f89e509cac0a69175bd4efb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-08T13:04:26Z (GMT). No. of bitstreams: 2 Diego Andres Velasco Acosta.pdf: 1957910 bytes, checksum: 57eabc538f89e509cac0a69175bd4efb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-01-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O balanço energético negativo (BEN) está associado com alterações na fertilidade de vacas leiteiras, portanto diferentes estratégias nutricionais e farmacológicas vêm sendo utilizadas para minimizar os efeitos negativos do BEN, visando aumentar a fertilidade. A aplicação pré-parto de somatotropina recombinante bovina (rbST) pode levar a uma redução da concentração sérica dos ácidos graxos não esterificados (AGNE) e β-hidroxibutirato (BHBA) no período pós-parto. Além disso têm sido demonstrado efeito significativo no desenvolvimento folicular ovariano pós-parto. Da mesma forma, estratégias nutricionais e o manejo da alimentação durante o período pré e pós-parto têm um impacto na saúde, produtividade e fertilidade da vaca leiteira. Estudos recentes mostram que a adição de aminoácidos na dieta durante o período de transição de vacas leiteiras pode ter efeitos benéficos no metabolismo, contribuindo para melhorar a performance reprodutiva. Diante desta problemática, esta tese apresenta três artigos, cujos objetivos foram: 1) determinar o efeito da administração pré-parto de somatotropina em novilhas leiteiras sobre marcadores do BEN, retomada da atividade ovariana e produção de leite. 2) Determinar o efeito da suplementação de metionina e colina protegidas da degradação ruminal, isoladas e em combinação, sobre o potencial esteroidogenico do folículo dominante da primeira onda folicular pós-parto de vacas leiteiras. 3) Determinar o efeito da suplementação de metionina e colina protegidas da degradação ruminal, isoladas e em combinação, sobre o desenvolvimento embrionário e nível de metilação global de DNA do embrião. O tratamento com somatotropina pré-parto em novilhas leiteiras aumentou a concentração de IGF-I intrafolicular e a expressão do receptor de LH e da proteína reguladora aguda da esteroidegênese em células foliculares do folículo dominante da primeira onda pós-parto. Essas mudanças foram associadas a um aumento da concentração de estradiol sérica e intrafolicular, o que pode, potencialmente, aumentar a chance de ovulação da primeira onda folicular pós-parto. Entretanto, somatotropina não afetou outros parâmetros metabólicos e produtivos no período pós-parto recente. A suplementação com metionina, colina ou ambas durante o período de transição parece não ter efeito sobre o desenvolvimento folicular pós-parto da primeira onda em vacas da raça Holandês. No entanto, a suplementação apenas com metionina durante o período de transição aumenta a expressão de 3β-HSD nas células foliculares. Além disso, a suplementação com metionina e colina induziu a uma down regulation dos genes pró-inflamatórios, indicando um menor processo inflamatório nas células foliculares. Em relação ao desenvolvimento embrionário, a suplementação com metionina durante o período de transição reduziu a metilação global do DNA e aumentou o acúmulo de lipídios nos embriões. A utilização de estratégias nutracêuticas durante o período de transição aumentaram o potencial esteroidogenico do folículo dominante da primeira onda folicular pós-parto e aumentou a expressão de genes relacionados a esteroidogenese, assim como uma redução na expressão de genes pró inflamatórios, indicando efeitos benéficos na fertilidade de bovinos leiteiros. Além disso, a suplementação de metionina parece aumentar a capacidade de sobrevivência dos embriões, desde que há fortes indícios de que as reservas lipídicas endógenas servem como um substrato energético. / The negative energy balance (NEB) is associated with changes in the fertility of dairy cows, therefore different nutritional and pharmacological strategies are being used to minimize the negative effects of the NEB in order to increase fertility. Pre-partum recombinant bovine somatotropin (rbST) administration can lead to a reduction in serum non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) in the postpartum period, in addition to improving postpartum ovarian follicular development. Similarly nutritional strategies during the pre and postpartum period have a positive impact on health, productivity and fertility of dairy cows. Recent studies show that the use of amino acids in the diet during the transition period may have beneficial effects in metabolism. Faced with this problem, this thesis presents three papers whosethe objectives were: 1) Determine the effect of pre-partum administration of somatotropin in dairy heifers on the markers of NEB, resumption of ovarian activity and milk production. 2) Determine the effect of methionine, choline supplementation isolated and in combination on esteroidogenic potential of the fist postpartum dominant follicle. 3) Determine the effect of methionine, choline supplementation isolated and in combination on embryo development and global DNA methylation. Pre-partum somatotropin treatment in dairy heifers increased intrafollicular IGF-I and mRNA expression of LH receptor and steroidogenic acute regulatory protein in follicular cells of the first postpartum dominant follicle. These changes were associated to increased intrafollicular and serum estradiol concentration, which can potentially increase the chance of ovulation of the first postpartum follicular wave. Despite that, somatotropin did not affect other metabolic and productive parameters in the early postpartum period. Supplementing methionine, choline or both during the transition period seemed to have no effect on postpartum follicular development of the first postpartum wave in Holstein cows. However, supplementing methionine, during the transition period increased follicular cells 3β-HSD expression, methionine and choline supplementation also induced a down regulation of pro-inflammatory genes, indicating a lower inflammatory processes in follicular cells. Regarding to embryo development, supplementation of methionine, only during the transition period reduce global DNA methylation, moreover, supplemented methionine increase lipid accumulation in preimplantation embryos. The use of nutraceutical strategies during the transition period increased the steroidogenic potential of the first postpartum dominant follicle and increase the expression of genes related steroidogenesis also a down regulation of proinflammatory genes indicating beneficial effects on fertility in dairy cattle. Moreover, the methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival since there is strong evidence that endogenous lipid reserves serve as an energy substrate.

Choline oxidase from Arthrobacter globiformis is a flavin-dependent enzyme that catalyzes the oxidation of choline to betaine aldehyde through two sequential hydride-transfer steps. The study of this enzyme is of importance to the understanding of glycine betaine biosynthesis found in pathogenic bacterial or economic relevant crop plants as a response to temperature and salt stress in adverse environment. In this study, chemical modification of choline oxidase using two irreversible inhibitors, tetranitromethane and phenylhydrazine, was performed in order to gain insights into the active site structure of the enzyme. Choline oxidase can also be inactivated irreversibly by freezing in 20 mM sodium phosphate and 20 mM sodium pyrophosphate at pH 6 and -20 oC. The results showed that enzyme inactivation was due to a localized conformational change associated with the ionization of a group in close proximity to the flavin cofactor and led to a complete lost of catalytic activity.

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, a limited number of compounds that accumulate to high levels in cytoplasm to prevent dehydration and plasmolysis in adverse hyperosmotic environments. With this respect, the study of choline oxidase has potential for the development of therapeutic agents that inhibit the biosynthesis of glycine betaine, thereby rendering pathogenic bacteria susceptible to either conventional treatments or the immune system. In this study, the highly GC rich codA gene encoding for choline oxidase was cloned, expressed. The resulting enzyme was purified to high levels, allowing for detailed biochemical, mechanistic and structural characterizations. A chemical mechanism for the reaction catalyzed by choline oxidase was established by using kinetic isotope effects and viscosity effects as probes, in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage. Furthermore, these experiments indicated that chemical steps of flavin reduction by choline and betaine aldehyde are rate limiting for the overall turnover of the enzyme. Further mechanistic characterization clearly suggested a hydride transfer mechanism that is fully quantum mechanical. The structure of choline oxidase was resolved at 1.86 Å resolution in collaboration with the group of Dr. Allen O. Orville, at the Georgia Institute of Technology, providing a structural framework that is consistent with the mechanistic studies. The results of these studies will be presented and discussed in the context of the Glucose-Methanol-Choline oxidoreductase enzyme superfamily, of which choline oxidase is a member. Previous structural and mechanistic studies of alcohol- and aldehyde-oxidizing enzymes with different cofactors, as well as the biotechnological and biomedical relevance of choline oxidase are presented in Chapter 1. Chapter 3-8 illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter 3), spectroscopic and steady state kinetics (Chapter 4), the determination of the chemical mechanism for alcohol oxidation and the investigation of the involvement of quantum mechanical tunneling (Chapter 5 and 6), the study of aldehyde oxidation (Chapter 7), and the structural determination of choline oxidase by x-ray crystallography (Chapter 8). Chapter 9 presents a general discussion of the data presented.

Choline oxidase catalyzes the oxidation of choline to glycine betaine. This reaction is of considerable medical and biotechnological applications, because the accumulation of glycine betaine in the cytoplasm of many plants and human pathogens enables them to counteract hyperosmotic environments. In this respect, the study of choline oxidase has potential for the development of a therapeutic agent that can specifically inhibit the formation of glycine betaine, and therefore render pathogens more susceptible to conventional treatment. The study of choline oxidase has also potential for the improvement of the stress resistance of plant by introducing an efficient biosynthetic pathway for glycine betaine in genetically engineered economically relevant crop plant. In this study, codA gene encoding for choline oxidase was cloned. The cloned gene was then used to express and purify the wild-type enzyme as well as to prepare selected mutant forms of choline oxidase. In all cases, the resulting enzymes were purified to high levels, allowing for detailed characterizations. The biophysical and biochemical analyses of choline oxidase variants in which the positively charged residue close to the flavin N(1) locus (His466) was removed (H466A) or reversed (H466D) suggest that in choline oxidase, His466 modulates the electrophilicity of the bound flavin and the polarity of the active site, and contributes to the flavinylation process of the covalently bound FAD as well as to the stabilization of the negative charges in the active site. Biochemical, structural, and mechanistic relevant properties of selected flavoproteins with special attention to flavoprotein oxidases, as well as the biotechnological and medical relevance of choline oxidase, are presented in Chapter I. Chapter II summarizes all the experimental techniques used in this study. Chapter III-VII illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter III), spectroscopic and steady state kinetics (Chapter IV), the catalytic roles of His466 and the effects of reversing the protein positive charge close to the flavin N(1) locus (Chapter V and VI), and the roles of His310 with a special attention to its involvement in a proton-transfer network (Chapter VII). Chapter VIII presents a general discussion of the data presented.