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3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase and 3-deoxy-Dmanno- octulosonate 8-phosphate synthase (KDO8P) synthase are two enzymes that catalyse very similar reactions. DAH7P synthase is the first enzyme of the shikimate pathway and catalyses the condensation reaction between the four-carbon sugar erythrose 4-phosphate (E4P) 1 and the three-carbon sugar phosphoenolpyruvate (PEP) 2 to give the seven-carbon sugar DAH7P 3. KDO8P synthase catalyses a similar condensation reaction between the five-carbon sugar arabinose 5-phosphate (A5P) 8 and PEP 2 to give the eight-carbon sugar KDO8P 9. Early mechanistic studies have shown the reaction mechanisms of these two enzymes to be very similar and structural and phylogenic analysis has suggested that the two enzymes share a common ancestor. However, there are differences between the two enzymes that have not been explained by the current literature. Whereas all DAH7P synthases require a divalent metal ion for activity, there exists both metallo and non-metallo KDO8P synthases. As well as this, there is the difference in substrate specificity. The natural substrate of KDO8P synthase, A5P, is one carbon longer and has the opposite C2 stereochemistry to E4P, the natural substrate of DAH7P synthase. This study investigates the role of the C2 and C3 hydroxyl groups of E4P and A5P in the enzyme catalysed reactions. The E4P analogues 2-deoxyE4P 38 and 3-deoxyE4P 39 have been synthesised from [beta]-hydroxy-[gamma]-butyrolactone and malic acid respectively. The two analogues were tested as substrates for DAH7P synthase from a variety of organisms, including N. meningitidis, the purification and characterisation of which was carried out during the course of these studies. It was found that both analogues were substrates for DAH7P synthase. 2-DeoxyE4P was found to be the best alternative substrate for DAH7P synthase to date. The analogous study was carried out on KDO8P synthase from N. meningitidis with 2- deoxyR5P 34 and 3-deoxyA5P 40. It was found that removal of the C2 and C3 hydroxyl groups of A5P was much more catastrophic for the KDO8P synthase catalysed reaction. Commercially available 2-deoxyR5P was found to be a very poor substrate, whereas 3-deoxyA5P, which was prepared according to a literature procedure was not a substrate. The difference in substrate specificities of DAH7P synthase and KDO8P synthase is consistent with the hypothesis that despite their similarities, these two related enzymes have different mechanisms. The key step for DAH7P synthase appears to be coordination of the E4P carbonyl to the divalent metal. The metal appears to play a less important role in the KDO8P synthase reaction and the key step is the correct orientation of A5P in the active site.

The genus Mycobacterium comprises some of the most devastating pathogens that infect humans. Mycobacterium tuberculosis causes tuberculosis in humans leading to high morbidity and mortality. The disease is especially prevalent in the under-developed and developing countries of the tropics. Diseases like AIDS and cancer compromise the immune system of an individual leaving him/her susceptible to secondary infections, particularly of tuberculosis. Thus, tuberculosis is making reappearance even in the well-developed countries of the west. The emergence of multi drug resistant strains of tuberculosis makes this deadly disease difficult to cure. A vaccine against tuberculosis is therefore the need of the hour. Mycobacterium smegmatis is a non-pathogenic member of the same family. It has a relatively fast multiplication time when compared to M. tuberculosis and shares the same unique features of the family that make pathogenic members extremely resistant to chemicals and drugs. Proteins of M. smegmatis and M. tuberculosis share high sequence identities, making M. smegmatis the microorganism of choice to study its more deadly counterpart from the same family. A striking feature of all mycobacterial genomes is the abundance of genes coding for enzymes involved in fatty acid and lipid metabolism; more than 250 in Mycobacterium tuberculosis compared to only 50 in Escherichia coli. The mycobacterial genome codes for over a hundred enzymes involved in fatty acid degradation. Apart from providing energy, lipids and fatty acids also form an integral part of the cell wall and cell membrane of Mycobacteria. The abundance and importance of lipid metabolizing enzymes in Mycobacteria make them attractive targets for drug discovery. It is therefore of interest to biochemically and structurally characterize these enzymes. Thiolases are a group of enzymes that are involved in lipid metabolism. In the last step of the β-oxidation pathway, degradative thiolases catalyze the shortening of fatty acid chains by degrading 3-keto acyl CoA to acetyl CoA and a shortened acyl CoA molecule. Thiolases are a subfamily of the thiolase superfamily. This superfamily also includes the Ketoacyl-(Acyl-carrier-protein)-Synthase (KAS) enzymes, polyketide synthases and chalcone synthases. Most members of this superfamily are dimers and while only a few have been found to be tetramers. The tetramers are loosely held dimers of tight dimers. Examination of the Mycobacterium smegmatis genome revealed the presence of several putative thiolase genes. These genes have been annotated as thiolases on the basis of sequence analysis. However, none of them has been biochemically or structurally characterized. The sequence identity between some of these proteins and the other well-characterized thiolases is rather low. The work described in this thesis attempts to characterize two such enzymes from M. smegmatis structurally and functionally. Chapter 1 begins with a brief introduction to the genus Mycobacteria and the role of fatty acid metabolism in mycobacterial virulence. This is followed by a review of the current literature on the enzymes of the thiolase superfamily and their role in fatty acid metabolism. The chapter concludes with a brief summary on the aims and objectives of the work. Chapter 2 describes all the common experimental procedures and computational methods used during the course of these investigations, as most of them are applicable to all the structure determinations and analyses presented in later chapters. The experimental procedures described include overexpression, purification, site directed mutagenesis, isolation of plasmids, crystallization of proteins and X-ray diffraction data collection. Computational methods include structure determination protocols along with details of various programs used during data processing, structure determination, refinement, model building, structure validation and analysis. Chapter 3 describes the cloning, expression, purification, crystallization and structure determination of a thiolase-like protein (TLP1) from M. smegmatis. All enzymes of the thiolase superfamily that have been structurally characterized so far share four features: 1) conservation of the core α/β/α/β/α-layered structure of the thiolase domain, 2) conservation of the extensive dimerization interface, 3) the location of the active site pocket and conservation of key active site residues and 4) the use of a nucleophilic cysteine residue in catalysis. The crystal structure of MsTLP1 revealed some interesting differences when compared to classical thiolases. Of the four characteristic features of thiolases, MsTLP1 has the conserved thiolase fold. The location of its putative active site is similar to that in classical thiolases. However, the dimerization is not a conserved feature in MsTLP1, which appears to be a monomer in solution as well as in the crystal structure. The ligand binding groove of MsTLP1, identified by structural superposition with Z. ramigera thiolase, is larger than that of Z. ramigera. The absence of the catalytic cysteine suggested that though the protein has the strictly conserved thiolase fold, it might perform an entirely different function. A unique extra C-terminal domain of unknown function present only in MsTLP1 has been described towards the end of the chapter. A thorough sequence and structural analysis suggested that MsTLP1 might belong to a new subfamily in the thiolase superfamily. Chapter 4 describes the attempts made towards the biochemical characterization of MsTLP1. Thiolase assays carried out for the synthetic and degradative reactions revealed that the enzyme is inactive in both the directions. However, surface plasmon resonance binding studies revealed that the protein could bind to Coenzyme A, a feature it shares with other enzymes of the thiolase superfamily. Thorough bioinformatics analyses of the structure to determine the residues involved in CoA binding have also been described. The chapter ends with a discussion on the probable function of TLPs in Mycobacteria. Chapter 5 describes the cloning, expression, purification and X-ray structural studies on MsT1-L thiolase. This is the first structural report of a probable T1-thiolase. The protein crystallized in three different space groups, in all of which the enzyme was found to be in a tetrameric form. Analysis of the tetramer structures from the three different crystal forms revealed that MsT1-L exhibits some rotational flexibility about the central tetramerization loop. A qualitative and quantitative analysis of this movement has been described. Structural comparisons revealed that the overall structure of MsT1-L is very similar to that of the well-characterized biosynthetic thiolase form Z. ramigera. However, a detailed analysis of the ordered waters near the active site cavity revealed interesting differences between the two. The probable functional relevance of this observation has been discussed. The crystal structure of MsT1-L complexed with CoA has also been described in detail. Structural comparisons with classical thiolases also revealed significant differences in the organization of the loop domain that harbors most of the residues required for catalysis. These differences cause the active site cavity of MsT1-L to be larger than that of biosynthetic thiolase suggesting that MsT1-L thiolase could probably bind larger substrates. This cavity is large enough to accommodate a medium chain length fatty acyl CoA as substrate. Co-crystallization experiments with hexanoyl CoA revealed a novel binding site for the fatty acyl chain in MsT1-L and this has been described in detail. Contributions made towards the cloning and expression of other thiolases from S. typhimurium and P. falciparum have been described in Chapters 6 and 7. The thesis concludes with a brief discussion on the future prospects of the investigations presented here.

The dissertation describes a novel method for plant drug discovery based on mutation and selection of plant cells. Despite the industry focus on chemical synthesis, plants remain a source of potent and complex bioactive metabolites. Many of these have evolved as defensive compounds targeted on key proteins in the CNS of herbivorous insects, for example the insect dopamine transporter (DAT). Because of homology with the human DAT protein some of these metabolites have high abuse potential, but others may be valuable in treating drug dependence. This dissertation redirects the evolution of a native Lobelia species toward metabolites with greater activity at this therapeutic target, i.e. the human DAT. This was achieved by expressing the human DAT protein in transgenic plant cells and selecting gain-of-function mutants for survival on medium containing a neurotoxin that is accumulated by the human DAT. This created a sub-population of mutants with increased DAT inhibitory activity. Some of the active metabolites in these mutants are novel (i.e. not detectable in wild-type cells). Others are cytoprotective, and also protect DAergic neurons against the neurotoxin. This provides proof-of-concept for a novel plant drug discovery platform, which is applicable to many different therapeutic target proteins and plant species.

Le repliement des protéines est principalement gouverné par les interactions spécifiques des structures secondaires. 1, 2 Toutefois, il existe expérimentalement peu d’informations sur les propriétés mécaniques fondamentales des hélices α et des feuillets β isolées. Les recherches antérieures sur l'étude du déploiement des hélices sont peu concluantes 3-5 et à notre connaissance l'étude des propriétés mécaniques d'un feuillet β isolé, intramoléculaire est sans précédent. Les copolymères PEG114-b-poly(L-lysine)134-(2-pyridyl disulfure),PEG114-b-poly(L-lysine)-b-PEG114 et poly(L-acide glutamique)85-b-(2-pyridyldisulfure) été synthétisés et utilisés comme systèmes modèles pour tester les propriétés mécaniques des motifs secondaires de type hélice α et feuillet β. Les résultats obtenus se sont révélés être en bon accord avec les résultats théoriques obtenus en utilisant un modèle statistique basé sur AGAGIR 6. La différence de force de déroulement comparant les hélices de poly(L-Lysine) ≈ 30 pN et de poly(L-acide glutamique) ≈ 20 pN des copolymères diblocs a été attribuée à l'hydrophobicité différente des chaînes latérales. La plus grande hydrophobie dumotif lysine conduit à de plus grandes interactions entre les chaînes latérales qui empêchent les fluctuations aléatoires au sein de l’hélice, et conduisent à une stabilité supérieure de l'hélice α. Lorsque les expériences ont été conduites dans des conditions favorisant la solubilité des chaînes latérales de lysine, les interactions ont diminué à une force de ≈ 20 pN, similaire à la force des interactions observées pour le poly(L-acide glutamique). Nous supposons qu'un minimum de ≈ 20 pN est nécessaire pour rompre la liaison hydrogène en maintenant l'hélice α, car cette force a été obtenue dans des conditions où les interactions de la chaîne latérale étaient minimisées. La présence de plateaux de force constants et d'inflexions correspondantes démontre une force de dépliement indépendante de la longueur, qui supporte un mécanisme de déroulement tour-par-tour pour l'hélice. De plus, la plus grande hydrophobie des chaînes latérales a été suggérée non seulement pour stabiliser la structure en hélice, mais également pour inhiber la formation d'une structure de type β-turn métastable intermédiaire lorsque les forces entropiques dominent. Des études préliminaires ont été effectuées sur le système de PEG114-bpoly(L-Lysine)134-(2-pyridyl disulfure) après induction d’une transition - β par un traitement thermique dans des conditions basiques. Une inflexion à une force≈ 70 pN a été obtenue, ce qui suggère la formation d'une interaction de type feuillet β. Une stratégie bottom-up a ainsi été proposée avec succès, démontrant le potentiel d'utilisation de tels systèmes artificiels pour simplifier et modéliser des systèmes biologiques réels. La compréhension de ces modèles isolés plus simples aidera sans doute la compréhension de systèmes plus complexes. / Proteins fold by the initial, preferential folding of secondarystructures 1, 2, however surprisingly little is known about the basic mechanicalproperties of isolated α-helices and β-sheets from an experimental standpoint.Previous investigations into studying the generic unfolding behaviour of α-heliceshave proved inconclusive 3-5, and to our knowledge the study of an isolated,intramolecular β-sheet is unprecedented.Bioinspired PEG114-b-poly(L-glutamic acid)85-(2-pyridyl disulphide),PEG114-b-poly(L-lysine)134-(2-pyridyl disulphide) and PEG114-b-poly(Llysine)134–b-PEG114 were designed, synthesized and utilized as model systems toprobe the mechanical properties of α-helix and β-sheet secondary motifs. Theobtained results were shown to be in good agreement with theoretical resultsobtained by utilizing a AGAGIR-based statistical mechanical model 6. Thedifference in unravelling force comparing the helices of poly(L-Lysine) ≈30 pNand poly(L-glutamic acid) ≈20 pN diblock copolymers was attributed to thediffering hydrophobicity of the side chains. The greater hydrophobicity of thelysine allowed greater interactions between the side chains and sterically hinderedrandom helix-coil fluctuations, which lead to a superior α-helix stability. Whenexperiments were conducted in conditions promoting the solubility of the lysineside chains, the interactions decreased to a force of ≈20 pN, similar to the force ofinteractions observed for the poly(L-glutamic acid). We infer that a minimum of≈20 pN is needed to rupture the hydrogen bonding maintaining the α-helix as thisforce was obtained in conditions where the side chain interactions wereminimized.The presence of constant force plateaus and corresponding inflectionsdemonstrates a length independent unfolding force, which supports a turn-by-turnunfolding mechanism for the α-helix.In addition, the greater hydrophobicity of the side chains was suggestedto not only stabilize the α-helix structure, but also to inhibit the formation of anintermediate metastable β-hairpin-like structure when entropic forces dominate.Preliminary studies were also conducted on the PEG114-b-poly(LLysine)134-(2-pyridyl disulphide) system after a α-β transition had been inducedby heat in basic conditions, where an inflection at a much higher force of ≈ 70 pNwas obtained suggesting the formation of a β-sheet interaction.A bottom-up, investigative strategy has thus been successfully proposeddemonstrating the potential of utilizing such artificial systems to simplify andexemplify real biological systems. The comprehension of these simpler isolatedmodels will no doubt aid the understanding of more complex systems.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

No description available.