The central dogma of molecular biology outlines the process of information transfer from a DNA sequence, to a protein chain. Beyond the step of protein synthesis, there are a variety of post-translational modifications that can take place, one of which is addition of carbohydrate chains to nascent proteins, known as glycosylation. The N-linked glycosylation pathway is responsible for the covalent attachment of multifunctional carbohydrate chains on asparagine residues of nascent proteins at Asn-X-Ser/Thr consensus sequences. These carbohydrate chains are thought to aid in cell signaling, immune recognition, and other processes. Golgi alpha-mannosidase II (GMII) is the enzyme in the N-glycosylation pathway that is responsible for cleaving two mannose linkages in the oligosaccharide GnMan5Gn2 (where Gn is N-acetylglucosamine and Man is mannose), thereby producing GnMan3Gn2 , which is the committed step in complex N-glycan synthesis. It has been speculated that GMII is an excellent therapeutic target for cancer treatment, as the unusual distribution of carbohydrates on the surface of tumour cells has been characterized in many cancers. In addition, swainsonine-—a strong, yet nonspecific inhibitor of GMII—-has been shown to block metastasis and improve the clinical outcome of patients with certain cancers, including those of the colon, breast and skin. This thesis examines Golgi alpha-mannosidase II from Drosophila melanogaster (dGMII) as a model for all GMII enzymes. First, a 1.80 Angstrom resolution crystal structure of a weak inhibitor, kifunensine, binding to dGMII provides mechanistic insights into the substrate distortion in the GMII reaction. It is hypothesized that the GMII reaction proceeds via a 1 Sinterintermedi-ate. Second, a 1.40 Angstrom resolution structure of a mutant dGMII bound to its natural substrate, GnMan5Gn, identifies key substrate binding and catalytic residues, as well as expanding the definition of the GMII active site to include two distant sugar−binding subsites. Finally, the results are taken together, with knowledge of other related enzymes to synthesize a plausible itinerary for the GMII reaction.

No description available.

No description available.

No description available.

The central dogma of molecular biology outlines the process of information transfer from a DNA sequence, to a protein chain. Beyond the step of protein synthesis, there are a variety of post-translational modifications that can take place, one of which is addition of carbohydrate chains to nascent proteins, known as glycosylation. The N-linked glycosylation pathway is responsible for the covalent attachment of multifunctional carbohydrate chains on asparagine residues of nascent proteins at Asn-X-Ser/Thr consensus sequences. These carbohydrate chains are thought to aid in cell signaling, immune recognition, and other processes. Golgi alpha-mannosidase II (GMII) is the enzyme in the N-glycosylation pathway that is responsible for cleaving two mannose linkages in the oligosaccharide GnMan5Gn2 (where Gn is N-acetylglucosamine and Man is mannose), thereby producing GnMan3Gn2 , which is the committed step in complex N-glycan synthesis. It has been speculated that GMII is an excellent therapeutic target for cancer treatment, as the unusual distribution of carbohydrates on the surface of tumour cells has been characterized in many cancers. In addition, swainsonine-—a strong, yet nonspecific inhibitor of GMII—-has been shown to block metastasis and improve the clinical outcome of patients with certain cancers, including those of the colon, breast and skin. This thesis examines Golgi alpha-mannosidase II from Drosophila melanogaster (dGMII) as a model for all GMII enzymes. First, a 1.80 Angstrom resolution crystal structure of a weak inhibitor, kifunensine, binding to dGMII provides mechanistic insights into the substrate distortion in the GMII reaction. It is hypothesized that the GMII reaction proceeds via a 1 Sinterintermedi-ate. Second, a 1.40 Angstrom resolution structure of a mutant dGMII bound to its natural substrate, GnMan5Gn, identifies key substrate binding and catalytic residues, as well as expanding the definition of the GMII active site to include two distant sugar−binding subsites. Finally, the results are taken together, with knowledge of other related enzymes to synthesize a plausible itinerary for the GMII reaction.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

Stuttgart, Univ., Diss., 2001.

Universiẗat, Diss., 2001--Stuttgart.

Les enzymes lipolytiques sont des ester hydrolases impliquées dans le métabolisme lipidique. Leurs caractéristiques se sont révélées être des atouts dans de nombreuses applications industrielles. Chez les microalgues, l’isolement et la caractérisation de ces enzymes d’un point de vue structural et fonctionnel restent des domaines de recherche peu explorés à ce jour.Certaines espèces bénéficient pourtant de contenus en lipides intéressants, source de matière première pour les industries de l’agroalimentaire ou de l’énergie. Par exemple, l’acide docosahexaénoique (DHA), un acide gras polyinsaturé de la série des omégas 3, est reconnu pour ses propriétés en santé humaine. Parmi de nombreuses espèces, Isochrysis galbana, une microalgue unicellulaire appartenant à la classe des Prymnesiophycées est considérée comme une source possible de DHA. La présence d’acides gras libres a été montrée par l’analyse des lipides, suggérant la présence d’enzymes lipolytiques potentiellement intéressantes pour leur sélectivité et leur spécificité de substrat.L’analyse d’une banque de marqueurs de séquences exprimées a permis l’identification de séquences susceptibles de coder des enzymes lipolytiques. Les ARN messagers ont été extraits et les ADN complémentaires ont été clonés.Ce travail de thèse présente l’analyse et le clonage de deux gènes codant une ester hydrolase putative et une thioestérase putative, issues de la microalgue Isochrysis galbana.Les deux séquences codent des protéines de poids moléculaires de 35,41 kDa et de 42,31 kDa. Elles montrent 30 à 40 % d’identité et de similarité avec des hydrolases notamment des carboxylestérasesde différents organismes. Les séquences protéiques ont permis l’identification du pentapeptide consensus Gly-X-Ser-X-Gly caractéristique des enzymes lipolytiques et les acides aminés Ser/Asp/His de la triade catalytique.Les deux séquences codantes ont été clonées et exprimées dans la levure Saccharomyces cerevisiae et la bactérie Escherichia coli. Le clonage dans E. coli a permis d’identifier à la taille attendue une protéine par Western blot. En présence de cette protéine, la composition en acides gras des lipides de la bactérie a été modifiée. L’analyse CPG a notamment montré une augmentation des proportions en acides gras C16 :1 et C18 :1 par rapport au témoin. Ce résultat permet de caractériser l’activité thioestérase pour IgTeCe. / Lipolytic enzymes present in all known species play a key role in lipid metabolism and are involved in several industrial processes. They catalyse lipid hydrolysis and synthesis. Actually and particularly in microalgae, isolation and characterization of this type of enzyme remains an unexplored research area.The potential of the lipidic content of microalgae in food industry or energy field requires specific lipolytic enzymes. Docosahexaenoic acid (DHA), an 3 poly insaturated fatty acid (3 PUFA) is well known for its beneficial effects on human health. Among many species, Isochrysis galbana, a unicellular marine microalga belonging to the Prymnesiophyceae class, is considered as a potential alternative source of DHA.Lipid analysis of I. galbana shows free fatty acids and suggests the presence of lipolytic enzymes with potential interesting selectivities and substrate specificities. Analysis of incomplete expressed sequence tag (EST) listed in the EST bank of Isochrysis galbana, identified incomplete genes that encode lipolytic enzymes. Messenger RNAs were extracted, characterized and cloned.This work describes the analysis and cloning of two genes encoding a putative ester hydrolase and a putative thioesterase in marine microalgae Isochrysis galbana. Sequences encode two proteins with predicted molecular weights of approximately 35,41 kDa and 42,31 kDa. Slight similarity and identity (from 30 to 40 %) were observed between the gene sequence and various  fold hydrolase found in diverse phyla (including carboxylesterase).Sequences also included the consensus Gly-X-Ser-X-Gly and the catalytic triad Ser/Asp/His. To characterize the predicted enzymatic functions, an experimental procedure was introduced: coding sequences were cloned into expression vectors and expressed in Saccharomyces cerevisiae and in Escherichia coli.Western blot identification of recombinant enzyme shows a convenient protein production in bacteria. Furthermore, the expression of the protein in E. coli shifted the fatty acid composition predominantly towards C16:1 and C18:1 fatty acids. The enzyme called IgTeCe showed a thioesterase activity.

abstract: Cocaine abuse affects millions of people with disastrous medical and societal consequences. Despite this, there is still no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts, and acute cocaine toxicity (overdose) is only symptomatically treated. Studies have demonstrated a promising potential treatment option with the help of the human serum enzyme butyrylcholinesterase (BChE), an enzyme capable of breaking down cocaine into biologically inactive side products. This activity of wild-type BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against cocaine. Plants were used as a sustainable, scalable, affordable platform system to produce large amounts of human biologics such as these cocaine hydrolase variants of BChE. Using a tobacco relative, Nicotiana benthamiana, recombinant enzymes can be produced at quantities relevant to clinical use with desired kinetic properties. Next, the ability of the most promising plant-produced cocaine super hydrolase, pCocSH, to counter the lethal effects of cocaine overdose in vivo was tested. These studies revealed that this plant-produced enzyme can protect mice from an otherwise lethal dose of cocaine. Most excitingly, it was found that pCocSH can rescue mice from overdose when given immediately after the onset of cocaine-induced seizures. These studies provide in vitro and in vivo proof-of-principle for a promising plant-derived biologic to be used as a pharmacokinetic-based treatment for cocaine addiction-related diseases such as overdose. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015