• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 101
  • 44
  • 21
  • 12
  • 8
  • 7
  • 7
  • 7
  • 7
  • 7
  • 7
  • 3
  • Tagged with
  • 231
  • 47
  • 44
  • 32
  • 26
  • 25
  • 22
  • 22
  • 21
  • 18
  • 18
  • 16
  • 15
  • 14
  • 14
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
121

Heme-dependent Tryptophan Oxidation: Mechanistic Studies on Tryptophan 2,3-Dioxygenase and MauG

Geng, Jiafeng 17 December 2014 (has links)
Hemoenzymes are prevalent in nature and participate in a wide range of biological activities. Frequently, high-valence iron intermediates are involved in the catalytic events of these enzymes, especially when the activation of peroxide or dioxygen is involved. Building on the fundamental framework of iron-oxygen chemistry, the mechanistic understandings of these enzymes and their reactive intermediates constantly attract attention from the enzymology community. This dissertation work focused on the mechanistic studies on two hemoenzymes, tryptophan 2,3-dioxygenase (TDO) and MauG, both of which catalyze unique chemical transformations, i.e., tryptophan oxidation. TDO and MauG are structurally distinct from each other; they catalyze different types of oxidization reactions on tryptophan via diverse strategies. TDO catalyzes the ring-cleavage dioxygenation reaction of free L-tryptophan, incorporating both oxygen atoms from dioxygen into the substrate. MauG uses hydrogen peroxide as the oxidant to catalyze a complex posttranslational-modification reaction on two tryptophan residues from a protein substrate. It utilizes radical chemistry to perform a 40-Å long-range catalytic event. Despite the differences in their catalytic behavior, both enzymes are suggested to employ high-valence iron intermediates in their reactions. A collection of biochemical and spectroscopic approaches was employed to obtain detailed insight into the electronic and structural contributions to the formation and stabilization of high-valence iron intermediates, and into the heme-dependent tryptophan-oxidizing mechanisms. In the study TDO, we solved the long-standing mystery of how the active Fe(II) enzyme is generated from the resting Fe(III) form by hydrogen peroxide. A peroxide-driven reactivation mechanism was established based on the identification of a compound ES-type ferryl intermediate. Additional efforts were dedicated to clarify the controversy in the literature regarding the catalytic roles of a distal histidine residue. Chemical-rescue approaches were used to specify the catalytic contributions of the target residue. In the study of MauG, we discovered an unprecedented tryptophan-mediated charge-resonance phenomenon in the bis-Fe(IV) redox state. This discovery provides the molecular basis for the chemical reactivity and stability of the catalytically competent bis-Fe(IV) intermediate. Together with our collaborators, we also outlined the mechanism of the MauG-mediated long-range catalysis by identifying the catalytic functions of several important residues along the reaction pathway.
122

Studies of enzyme kinetics and aspects of enzyme structure in vivo using NMR and molecular genetics

Williams, Simon-Peter January 1992 (has links)
A quantitative understanding of metabolic control depends on a knowledge of the enzymes involved. The extrapolation of studies in vitro to the intact cell is controversial because the intracellular environment is relatively poorly characterised, particularly with respect to the interactions between weakly-associated enzymes. There is a clear need to study enzymes directly in the cell, yet there are few suitable techniques. Metabolites have been very successfully studied in cells by the non-invasive technique of nuclear magnetic resonance (NMR). NMR studies of enzymes in the cell have, however, been prevented by difficulties in assigning the resonances from the many proteins within the cell. A method for studying a specific enzyme in the cell has been developed, using Saccharomyces cerevisiae and phosphoglycerate kinase (PGK) as a model system. Using an inducible expression system, PGK was synthesised in the cell without significant synthesis of other proteins. With 5-fluorotryptophan in the growth medium, fluorine-labelled PGK was formed in situ. Fluorine is an excellent label for NMR since it is absent from most cells and has a high receptivity to NMR detection. <sup>19</sup> F NMR was used to study PGK in the intact cell. Comparisons with measurements in vitro showed that PGK was exposed to only a small fraction of the total intracellular [ADP], implying some form of compartmentalisation. The NMR relaxation properties observed in vivo and in vitro were compared with theoretical predictions. This showed that PGK was not part of a complex in the cell and that the viscosity of the cytoplasm, relative to water, was c. 4 at 30 °C. Fluorine-labelled pyruvate kinase and hexokinase have also been prepared; the spectra of these proteins in vitro are responsive to their ligands, and further work will study these proteins in vivo. NMR techniques were also applied to study the kinetics of PGK in the cell. PGK and GAPDH catalyse an ATP↔P<sub>i</sub> exchange which is near-equilibrium in wild-type cells. <sup>31</sup>P magnetisation transfer experiments in genetically manipulated cells showed that the reaction becomes unidirectional if the PGK activity is reduced by 95 %. Net flux is reduced by less than 30 %. In low-PGK cells, the ATP↔P<sub>i</sub> exchange from oxidative phosphorylation can be isolated from that of glycolysis, facilitating direct measurements of the P:O ratio. In the cells studied, the P:O ratio was 2 to 3.
123

Cholinesterase inhibitors in Alzheimer's disease : an experimental study on mechanisms of interaction with muscarinic and nicotinic receptors and neuroprotection /

Svensson, Anne-Lie, January 1900 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.
124

Structure and dynamics of the receptor kinase interacting FHA domain of kinase associated protein kinase from arabidopsis

Lee, Gui-in, January 2003 (has links)
Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "August 2003." Typescript. Vita. Includes bibliographical references (leaves 158-174). Also issued on the Internet.
125

Mechanistic and Structural Characterization of Thiamine Diphosphate Dependent Enzyme Transketolases from Human and E.coli

Dai, Shao-Bo 20 June 2017 (has links)
No description available.
126

Estudos funcionais e moleculares relativos às membranas apicais intestinais de Dysdercus peruvianus / Functional and molecular studies related to apical midgut membranes of Dysdercus peruvianus

André Coppe Pimentel 12 August 2016 (has links)
Os insetos da ordem Hemiptera apresentam membranas lipoproteicas que revestem as microvilosidades das células intestinais, como se fossem dedos de luva, e formam expansões para o lúmen do intestino que parecem terminar em fundo cego. A presença das duas membranas no ápice dos enterócitos gera questões intrigantes de como se dá a formação da membrana perimicrovilar, como ocorre a absorção de nutrientes e como se dá o encaminhamento de enzimas digestivas para o lúmen intestinal. A digestão de proteínas baseada em enzimas originalmente lisossômicas é uma característica marcante nos Hemiptera, em especial os Heteroptera que evolutivamente voltaram a uma alimentação de polímeros. O presente estudo indica que os genes das proteinases tipicamente lisossômicas sofrem uma série de duplicações, havendo a manutenção de um gene para função puramente lisossômica e a divergência funcional dos demais genes para a função de digestão extracelular. O gene que mantém a função lisossômica não é modulado pela alimentação, além de ser expresso nos mais diversos tecidos. Já os genes que se especializaram na digestão extracelular têm a expressão aumentada com a ingestão de alimento, indicando sua função. Parece não haver diferença nas características relacionadas ao encaminhamento celular das proteínas produzidas por esses genes, indicando que o direcionamento para a rota secretória é devido à superexpressão dos genes relacionados à digestão. Enzimas como alfa-glicosidases, alfa-manosidases e aminopeptidases que participam da digestão extracelular seguem a rota secretória que envolve a formação de vesículas de dupla membrana. Foi possível ampliar o modelo de digestão incluindo a participação das catepsinas D, de uma alfa-glicosidase solúvel e a possível participação de uma tiolredutase além do definir o local de atuação das lipases. Temos agora uma visão global da participação das enzimas digestivas que atuam na digestão em D. peruvianus. / The insects of the order Hemiptera have lipoprotein membranes lining the microvilli of midgut cells, like glove fingers, and form expansions into the lumen of the intestine. The presence of two membranes on the apex of enterocytes thus generates intriguing questions about the formation of perimicrovillar membrane, the absorption of nutrients, and the targeting of digestive enzymes into the intestinal lumen. The digestion of proteins based on originally lysosomal enzymes is an important feature in Hemiptera, especially in Heteroptera that evolutionary returned to feed on polymers. The genes of typical lysosomal proteinases undergo a series of duplications followed by the maintenance of a gene for purely lysosomal function and functional divergence of other genes for extracellular digestion function. The gene that maintains the lysosomal function is not modulated by feeding, in addition to being expressed in diverse tissues. By the other hand, genes specialized in extracellular digestion are up regulated by food intake, indicating its function. No difference was found in the targeting in the proteins produced by these genes, which indicates that targeting to the secretory route is due to overexpression of digestion-related genes. Enzymes involved in extracellular digestion as alpha-glucosidase, alpha-mannosidase and aminopeptidase follow the secretory route that includes the formation of double membrane vesicles. In this study we increased the digestion model adding the participation of cathepsin D, a soluble alpha-glucosidase, and the possible participation of a tiolredutase, and also to defining the place of lipases operation. We now have a global view of the participation of digestive enzymes involved in digestion D. peruvianus.
127

Diversidade do potencial amilolítico em fungos filamentosos: purificação e caracterização de uma glucoamilase de Aspergillus brasiliensis / Diversity of amylolytic potential in filamentous fungi: purification and characterization of a glucoamylase from Aspergillus brasiliensis

Paula Zaghetto de Almeida 29 April 2015 (has links)
O Brasil apresenta cerca de 10 a 17,6% da biodiversidade mundial e apenas uma fração dela é conhecida. Os fungos filamentosos são bons produtores de enzimas e despertam um grande interesse biotecnológico. O amido é o principal carboidrato de reserva das plantas. Dentre as enzimas amilolíticas estão as glucoamilases, que catalisam a hidrólise das ligações -1,4 e -1,6 das extremidades da cadeia do amido liberando glucose. Neste trabalho foram isolados 25 fungos filamentosos de amostras de materiais em decomposição da Mata Atlântica. Dos micro-organismos com alta atividade amilolítica foram selecionados e identificados Aspergillus brasiliensis e Rhizopus oryzae. Foi realizada a otimização do cultivo e caracterização das amilases do extrato bruto de ambos os fungos. Após a obtenção destes dados foi selecionado A. brasiliensis, pois, sua amilase é mais termoestável e ainda não reportada na literatura. Após purificação a enzima foi identificada como glucoamilase, a qual é monomérica com 69 kDa e contém aproximadamente 21% de carboidratos. Apresenta um domínio de ligação ao amido na porção terminal e estrutura secundária rica em -hélice. Sua atividade ótima ocorre em pH 4,5 a 60°C, seu pI é de 3,21, pode ser ativada com a adição de Mn2+, e é inibida por glucose em concentrações maiores que 0,1 M. A glucoamilase apresenta excelente estabilidade ao pH e boa estabilidade a temperatura (a 50°C mantém 67% de atividade após 7 horas; a 55°C a meia vida é de 147 minutos). Com amido de batata a enzima apresentou as seguintes constantes cinéticas (km 2,21 mg/mL; Vmáx 155 U/mg; kcat 179 s-1; kcat/km 81,06). A glucoamilase foi imobilizada em DEAE-PEG com ativação de 12 vezes e possibilidade de reuso de 10 vezes com perda de apenas 31% de atividade. O derivado demostrou maior facilidade para hidrolisar a amilopectina do que à amilose. Também foi realizada uma análise de neighbor joining, que agrupou a glucoamilase de A. brasiliensis próxima às glucoamilases de espécies de Aspergillus, que são consideradas as mais derivadas. / Brazil holds about 10-17.6% of the world\'s biodiversity and just a percentage of it is known. Filamentous fungi are enzyme producers that have great biotechnological application. Starch is the main reserve carbohydrate in plants. Among the amylolytic enzymes there are the glucoamylases, that catalyze the hydrolysis of -1,4 and -1,6 linkages of the end of starch chains, and releases glucose. In this research 25 filamentous fungi from Atlantic forest decaying material samples were isolated. Among microorganisms with high amylolytic activity Aspergillus brasiliensis and Rhizoupus oryzae were selected and identified. The cultivation parameters were optimized and the enzymes of crude extract were characterized. Considering the previous data Aspergillus brasiliensis was selected because its amylases are more thermostable and it has not been described in the literature yet. After purification the enzyme was identified as a glucoamylase, which is monomeric with 69 kDa and about 21% of carbohydrates in its composition. The enzyme has a starch binding domain in the terminal position and its secondary structure is rich in -helix. The optimum pH for glucoamylase activity is 4.5, the temperature is 60ºC and its pI is 3.21. The enzyme can be activated by the addition of Mn+2, and inhibited in concentrations above 0,1M glucose. The glucoamylase has an excellent pH stability and a good temperature stability (at 50ºC 67% of the activity was retained after 7 hours; at 55°C its half-life was 147 minutes). The best kinetic values were obtained with potato starch (km 2.21 mg/mL; Vmax 155 U/mg; kcat 179 s-1; kcat/km 81,06). The glucoamylase was immobilized on DEAE-PEG, with an activation of 12 times and enzyme reuse 10 times with just 31% loss of its activity. The immobilized enzyme has a greater activity on amylopectin than amylose. A neighbor joining analysis with glucoamylases from filamentous fungi species was made and Aspergillus brasiliensis glucoamylase was grouped close to the glucoamylases of Aspergillus species, which are considered the most derivative.
128

Structural and Functional characterization of flavoenzymes involved in posttranscriptional modification of tRNA / Caractérisation structurale et fonctionnelle de flavoenzymes impliquées dans la modification posttranscriptionnelle des acides ribonucléiques de transfert

Bou Nader, Charles 28 September 2017 (has links)
La modification posttranscriptionnelle des acides ribonucléiques (ARNs) est une étape de maturation conservée dans tous les domaines du vivant. Mes travaux de thèse ont porté sur la caractérisation fonctionnelle et structurale de flavoenzymes impliquées dans la modification des ARN de transfert (ARNt) : les dihydrouridines synthases (Dus) dictant la formation de dihydrouridine via la flavine mononucléotide (FMN) et TrmFO responsable de la méthylation en C5 de l'uridine 54 via la flavine adénosine dinucléotide (FAD) ainsi que le methylènetétrahydrofolate. Afin d'élucider le mécanisme de TrmFO, nous avons élaboré une apoenzyme grâce à une simple mutation qui est efficacement reconstituée in vitro. Nous avons chimiquement synthétisé l'intermédiaire catalytique qui consiste en un FAD-iminium comportant un methylène sur le N5 de l'isoalloxazine. Cette espèce synthétique a été caractérisée par spectrométrie de masse et absorption UV-visible. La reconstitution de TrmFO avec cette molécule restore l'activité in vitro sur un ARNt transcrit prouvant le rôle du FAD comme agent méthylant via une méthylation réductrice. Dus2 réduit spécifiquement U20 et est constituée d'un Dus domaine néanmoins, chez les mammifères un double-stranded RNA-binding domaine (dsRBD) est présent. Afin de comprendre la fonction de cette organisation modulaire, nous avons montré que seule l'enzyme sauvage est active contrairement aux domaines isolés. Nous avons résolu les structures cristallographiques des deux domaines suggérant une redistribution des charges positives en surface. Ce dsRBD dicte la reconnaissance de l'ARNt en se fixant à la tige acceptrice/Tpsi. Ceci est régulé par une extension N-terminal, mis en évidence par des mutations, des titrations RMN ainsi qu’une structure cristallographique en complexe avec un ARN de 22 nucléotides. Ce travail illustre l’acquisition d’un dsRBD au cours de l’évolution dont la fonction est étendu à la reconnaissance des ARNts. / Posttranscriptional modification of ribonucleic acids (RNAs) is a crucial maturation step conserved in all domains of life. During my thesis, I have brought structural and functional insights on flavoenzymes involved in transfer RNA (tRNA) modifications: dihydrouridine synthase (Dus) responsible for dihydrouridine formation using flavin mononucleotide (FMN) and TrmFO responsible for C5 methylation of uridine position 54 relying on flavin adenosine dinucleotide (FAD) and methylenetetrahydrofolate. To elucidate the chemical mechanism of TrmFO we designed an apoprotein via a single mutation that could be reconstituted in vitro with FAD. Furthermore, we chemically synthesized the postulated intermediate active species consisting of a flavin iminium harboring a methylene moiety on the isoalloxazine N5 that was further characterized by mass spectrometry and UV-visible spectroscopy. Reconstitution of TrmFO with this molecule restored in vitro activity on a tRNA transcript proving that TrmFO uses FAD as a methylating agent via a reductive methylation.Dus2 reduces U20 and is comprised of a canonical Dus domain however, mammals have an additional double-stranded RNA-binding domain (dsRBD). To bring functional insight for this modular organization, we showed that only full length human Dus2 was active while its isolated domains were not. tRNA recognition is driven by the dsRBD via binding the acceptor and TΨ stem of tRNA with higher affinity then dsRNA as evidenced by NMR. We further solved the X-ray structures for both domains showing redistribution of surface positive charges justifying the involvement of this dsRBD for tRNA recognition in mammalian Dus2. This was attributed to a peculiar N-terminal extension proven by mutational analysis and an X-ray structure of dsRBD in complex with 22-nucleotide dsRNA. Altogether our work illustrates how during evolution, Dus2 enzymes acquired an engineered dsRBD for efficient tRNA binding via a ruler mechanism.
129

Caractérisation biochimique et biophysique des deux cytidylyltransférases de Plasmodium falciparum, enzymes clés du métabolisme des phospholipides / Biochemical and biophysical characterization of the two Plasmodium falciparum cytidylyltransferases, key enzymes of the malaria phospholipid metabolism

Contet, Alicia 06 May 2015 (has links)
Le paludisme est causé par l'infection et la destruction des érythrocytes par les parasites protozoaires appartenant au genre Plasmodium. Au cours de son développement dans l'érythrocyte,Plasmodium falciparum requiert la biosynthèse massive de membranes dont les principaux constituants lipidiques sont des phospholipides. La phosphatidylcholine (PC) et la phosphatidyléthanolamine (PE) représentent à elles deux environ 80 % des lipides membranaires et l'inhibition de leur biosynthèse est létale pour le parasite. La PC et la PE sont synthétisées par le parasite, principalement via les voies de novo dépendantes de la CDP-choline et de la CDP-éthanolamine (ou voies de Kennedy) en utilisant respectivement la choline et l'éthanolamine comme précurseurs. Ces travaux de thèse se focalisent sur les deux enzymes CTP:phosphocholine etCTP:phosphoéthanolamine cytidylyltransférase (PfCCT et PfECT, respectivement), catalysant les étapes limitantes des voies de Kennedy. Chez Plasmodium, les CCT et ECT possèdent deux domaines cytidylyltransférases (CT) portant l'activité catalytique, séparés par une longue région de liaison. Pour la CCT, cette duplication est retrouvée seulement chez trois organismes, tous faisant partie du phylumdes Apicomplexes : Babesia, Theileria et Plasmodium, alors que la présence de deux domaines CT estune caractéristique retrouvée chez toutes les ECT étudiées à ce jour. La première partie de ce travail de thèse concerne la caractérisation biochimique et l'inhibition la PfCCT Nous avons montré que les deux domaines CT de la PfCCT sont actifs à l'inverse de la PfECT pour laquelle seul le domaine CTN-terminal est catalytiquement actif. A la suite d'un criblage virtuel basé sur la structure de l'enzyme,nous avons identifié un composé princeps capable d'inhiber l'activité de la PfCCT in vitro, la synthèse de PC et la croissance parasitaire. Ce premier composé actif (haut µM) représente une base pour l'optimisation future de nouveaux composés plus efficaces. Dans la deuxième partie de cette thèse,nous avons déterminé le mécanisme catalytique, la spécificité de liaison des ligands et l'organisation structurale de la PfECT grâce à la combinaison d'approches biochimiques et biophysiques. L'ensemble des résultats présentés dans ce manuscrit apportent un éclairage important concernant le fonctionnement de ces deux cibles potentielles et constituent des étapes essentielles à l'élaboration d'une approche thérapeutique. / Malaria is caused by the infection and destruction of red blood cells by protozoan parasitesbelonging to the genus Plasmodium. During its intra-erythrocytic development, Plasmodiumfalciparum requires massive biosynthesis of membranes which are mainly composed of phospholipids.Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) together represent about 80% of thetotal membrane lipids and inhibition of their biosynthesis leads to parasite death. PC and PE aresynthesized by the parasite's machinery mainly through the de novo CDP-choline and CDPethanolamine(Kennedy) pathways using respectively choline and ethanolamine as precursors. Thisstudy focuses on the rate limiting steps of these pathways catalyzed by CTP:phosphocholine andCTP:phosphoethanolamine cytidylytransferases (PfCCT and PfECT, respectively). In Plasmodiumspecies, both CCT and ECT contain two catalytic cores (CT domains) separated by a long linker.Interestingly, for CCT this feature is found only in three organisms, all from the phylum ofApicomplexa: Babesia, Theileria and Plasmodium, whereas the presence of two CT domains is ageneral feature in all ECTs known so far. The first part of this work consists in the biochemicalcharacterization of PfCCT and the investigation of its druggability. We showed that both PfCCT CTdomains are active and display similar kinetic parameters while only the N-terminal CT domain wasactive in PfECT. Subsequent to an in silico structure-based screening of compounds libraries, weidentified a PfCCT inhibitor able to inhibit PC synthesis as well as P. falciparum growth in vitro in thehigh µM range. This compound represents a first step toward the optimization of future more potentcompounds. In the second part of this study, we investigated the catalytic mechanism of PfECT anddeciphered its interactions with its ligands using biochemical, biophysical and structural approaches.Collectively, these results bring new insights into the biochemical and structural properties of thesetwo keys enzymes of the phospholipid metabolism in P. falciparum and pave the way for their futuredevelopment as potential drug target.
130

Evolvability of a viral protease : experimental evolution of catalysis, robustness and specificity

Shafee, Thomas January 2014 (has links)
The aim of this thesis is to investigate aspects of molecular evolution and enzyme engineering using the experimental evolution of Tobacco Etch Virus cysteine protease (TEV) as a model. I map key features of the local fitness landscape and characterise how they affect details of enzyme evolution. In order to investigate the evolution of core active site machinery, I mutated the nucleophile of TEV to serine. The differing chemical properties of oxygen and sulphur force the enzyme into a fitness valley with a >104-fold activity reduction. Nevertheless, directed evolution was able to recover function, resulting in an enzyme able to utilise either nucleophile. High-throughput screening and sequencing revealed how the array of possible beneficial mutations changes as the enzyme evolves. Potential adaptive mutations are abundant at each step along the evolutionary trajectory, enriched around the active site periphery. It is currently unclear how seemingly neutral mutations affect further adaptive evolution. I used high-throughput directed evolution to accumulate neutral variation in large, evolving enzyme populations and deep sequencing to reconstruct the complex evolutionary dynamics within the lineages. Specifically I was able to observe the emergence of robust enzymes with improved mutation tolerance whose descendants overtake later populations. Lastly, I investigate how evolvability towards new substrate specificities changed along these neutral lineages, dissecting the different determinants of immediate and long-term evolvability. Results demonstrate the utility of evolutionary understanding to protease engineering. Together, these experiments forward our understanding of the molecular details of both fundamental evolution and enzyme engineering.

Page generated in 0.0471 seconds