Academic literature on the topic 'Tlp15'

Toll-like receptors (TLRs) play important role in the innate immune system. TLR15 is reported to have a unique role in defense against pathogens, but its structural and evolution characterizations are still poorly understood. In this study, we identified 57 completed TLR15 genes from avian and reptilian genomes. TLR15 clustered into an individual clade and was closely related to family 1 on the phylogenetic tree. Unlike the TLRs in family 1 with the broken asparagine ladders in the middle, TLR15 ectodomain had an intact asparagine ladder that is critical to maintain the overall shape of ectodomain. The conservation analysis found that TLR15 ectodomain had a highly evolutionarily conserved region on the convex surface of LRR11 module, which is probably involved in TLR15 activation process. Furthermore, the protein–protein docking analysis indicated that TLR15 TIR domains have the potential to form homodimers, the predicted interaction interface of TIR dimer was formed mainly by residues from the BB-loops andαC-helixes. Although TLR15 mainly underwent purifying selection, we detected 27 sites under positive selection for TLR15, 24 of which are located on its ectodomain. Our observations suggest the structural features of TLR15 which may be relevant to its function, but which requires further experimental validation.

Background: Lactoferricin peptide (LP) has been reported to control cancer cell proliferation. NF-κB interacting lncRNA (NKILA) is a tumor suppressor in several cancers. Objective: We aimed to explore the potential function of the truncated LP (TLP) in the prevention of cervical cancer cell proliferation. Methods: Bioinformatics analysis via PPA-Pred2 showed that 18-aa N-terminus of truncated lactoferricin peptide (TLP18, FKCRRWQWRMKKLGAPSI) shows higher affinity with nuclear factor kappaB (NF-κB) than LP. The effects of LP and TLP18 on cervical cancer cells SiHa and HeLa and the related mechanisms were explored by investigating NF-κB and lncRNA-NKILA. Results: TLP18 shows an inhibitory rate up to 0.4-fold higher than LP on the growth of cervical cancer cells (P<0.05). NKILA siRNA promoted cell growth whether LP or TLP18 treatment (P<0.05). TLP18 treatment increases the level of lncRNA-NKILA and reduces the level of NF-κB up to 0.2-fold and 0.6-fold higher than LP (P<0.05), respectively. NKILA siRNA increased the levels of NF-κB, cleaved caspase-3, and BAX (P<0.05). TLP18 increased apoptotic cell rate up to 0.2-fold higher than LP, while NKILA siRNA inhibited cell apoptosis cell growth even LP or TLP18 treatment. Conclusion: Truncated Lactoferricin peptide controls cervical cancer cell proliferation via lncRNA- NKILA/NF-κB feedback loop.

Campylobacter jejuni is a bacterial pathogen that is a common cause of enteritis in humans. We identified a previously uncharacterized type of sensory domain in the periplasmic region of the C. jejuni chemoreceptor Tlp10, termed the DAHL domain, that is predicted to have a bimodular helical architecture. Through two independent ligand-binding sites in this domain, Tlp10 responded to molecular aspartate, isoleucine, fumarate, malate, fucose, and mannose as attractants and to arginine, galactose, and thiamine as repellents. Tlp10 also recognized glycan ligands when present as terminal and intermediate residues of complex structures, such as the fucosylated human ganglioside GM1 and Lewisa antigen. A tlp10 mutant strain lacking the ligand-binding sites was attenuated in its ability to colonize avian caeca and to adhere to cultured human intestinal cells, indicating the potential involvement of the DAHL domain in host colonization and disease. The Tlp10 intracellular signaling domain interacted with the scaffolding proteins CheV and CheW, which couple chemoreceptors to intracellular signaling machinery, and with the signaling domains of other chemoreceptors, suggesting a key role for Tlp10 in signal transduction and incorporation into sensory arrays. We identified the DAHL domain in other bacterial signal transduction proteins, including the essential virulence induction protein VirA from the plant pathogen Agrobacterium tumefaciens. Together, these results suggest a potential link between Tlp10 and C. jejuni virulence.

ABSTRACT Toll-like receptors (TLRs) are a group of highly conserved molecules that initiate the innate immune response to pathogens by recognizing structural motifs expressed by microbes. We have identified a novel TLR, TLR15, by bioinformatic analysis of the chicken genome, which is distinct from any known vertebrate TLR and thus appears to be avian specific. The gene for TLR15 was sequenced and is found on chromosome 3, and it has archetypal TIR and transmembrane domains and a distinctive arrangement of extracellular leucine-rich regions. mRNA for TLR15 was detected in the spleen, bursa, and bone marrow of healthy chickens, suggesting a role for this novel receptor in constitutive host defense. Following in vivo Salmonella enterica serovar Typhimurium infection, quantitative real-time PCR demonstrated significant upregulation of TLR15 in the cecum of infected chickens. Interestingly, similar induction of TLR2 expression following infection was also observed. In vitro studies revealed TLR15 upregulation in chicken embryonic fibroblasts stimulated with heat-killed S. enterica serovar Typhimurium. Collectively, these results suggest a role for the TLR in avian defense against bacterial infection. We hypothesize that TLR15 may represent an avian-specific TLR that has been either retained in chicken and lost in other taxa or gained in the chicken.

ABSTRACT Motility responses triggered by changes in the electron transport system are collectively known as energy taxis. In Azospirillum brasilense, energy taxis was shown to be the principal form of locomotor control. In the present study, we have identified a novel chemoreceptor-like protein, named Tlp1, which serves as an energy taxis transducer. The Tlp1 protein is predicted to have an N-terminal periplasmic region and a cytoplasmic C-terminal signaling module homologous to those of other chemoreceptors. The predicted periplasmic region of Tlp1 comprises a conserved domain that is found in two types of microbial sensory receptors: chemotaxis transducers and histidine kinases. However, the function of this domain is currently unknown. We characterized the behavior of a tlp1 mutant by a series of spatial and temporal gradient assays. The tlp1 mutant is deficient in (i) chemotaxis to several rapidly oxidizable substrates, (ii) taxis to terminal electron acceptors (oxygen and nitrate), and (iii) redox taxis. Taken together, the data strongly suggest that Tlp1 mediates energy taxis in A. brasilense. Using qualitative and quantitative assays, we have also demonstrated that the tlp1 mutant is impaired in colonization of plant roots. This finding supports the hypothesis that energy taxis and therefore bacterial metabolism might be key factors in determining host specificity in Azospirillum-grass associations.

This study explored the potential impacts of cricket-derived chitin and chitosan on the immune systems of Cobb500 broilers. One hundred and fifty broiler chicks of the Cobb500 strain were randomly assigned to any one of the five dietary groups in order to accomplish this. While the first set of birds (group 1) were only served the basal diet with no supplementation, the second to fifth sets of birds (groups 2 to 5) were served a diet supplemented with 500 mg/kg of the following: cricket-chitin, cricket-chitosan, shrimp-chitin, and shrimp-chitosan. The bursa and spleen were weighed relative to the body weight, and qPCR was used to determine the spleen's relative expression of toll-like receptor 4 (TLR4), toll-like receptor 15 (TLR15), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) genes. After 42 days of dietary cricket-chitin, the bulk of the index immunological organs increased (P<0.05). At day 21, TLR4, TLR15, IL-1β, and iNOS expression were unaffected by chitin and chitosan, but at day 42, they were down-regulated (P<0.05). However, during day 21, dietary shrimp-derived chitosan enhanced (P<0.05) the relative expression of TLR4, TLR15, and IL-1β, whereas the expression of TLR15 was lowered (P<0.05) but that of TLR4 was increased by cricket-chitin and shrimp-chitin. According to our findings, feeding broiler chicks with 500 mg/kg of shrimp chitosan and cricket-derived chitin can positively boost their immunity.

El objetivo principal fue evaluar el mecanismo de RSI en “Thompson Seedless”, al estímulo de los genes (Lox2, Tlp1, Npr1, Eir1). Se verifico la activación de genes de defensa para hojas y raíces, demostrando la activación sistémica por inoculación de P. veronii R4 en ‘Thompson Seedless’. Mediante q-PCR observamos la expresión del gen Lox2 en hojas de vid, con aumento progresivo hasta (12 h de post inoculación) de muestreo, al analizar este mismo gen en raíces no existió estímulo alguno. Los niveles transcripcionales de los genes Eir1 y Tlp1, fueron estimulados solo en raíces al ser expuestas por R4. Los niveles de estímulo del gen Tlp1 se relaciona con la habilidad que tiene R4 en desarrollar el complejo de simbiosis inducido por la vía del etileno (ET). Los niveles de estímulo del gen Npr1 fueron constitutivos en hojas y raíces, no encontrando diferencias signi cativas entre plantas tratadas con PBS o R4. Los resultados demuestran que R4, en contacto con raíces de ‘Thompson Seedless’, estimulan la expresión de los genes Eir1, Lox2, Tlp1 a 5 min, 6 y 12 h de post inoculación, permitiendo establecer la efectividad de RSI dirigida por la cepa R4 en “Thompson Seedless” con riendo un estado de prealerta.

El objetivo principal fue evaluar el mecanismo de RSI en “Thompson Seedless”, al estímulo de los genes (Lox2, Tlp1, Npr1, Eir1). Se verifico la activación de genes de defensa para hojas y raíces, demostrando la activación sistémica por inoculación de P. veronii R4 en ‘Thompson Seedless’. Mediante q-PCR observamos la expresión del gen Lox2 en hojas de vid, con aumento progresivo hasta (12 h de post inoculación) de muestreo, al analizar este mismo gen en raíces no existió estímulo alguno. Los niveles transcripcionales de los genes Eir1 y Tlp1, fueron estimulados solo en raíces al ser expuestas por R4. Los niveles de estímulo del gen Tlp1 se relaciona con la habilidad que tiene R4 en desarrollar el complejo de simbiosis inducido por la vía del etileno (ET). Los niveles de estímulo del gen Npr1 fueron constitutivos en hojas y raíces, no encontrando diferencias signi cativas entre plantas tratadas con PBS o R4. Los resultados demuestran que R4, en contacto con raíces de ‘Thompson Seedless’, estimulan la expresión de los genes Eir1, Lox2, Tlp1 a 5 min, 6 y 12 h de post inoculación, permitiendo establecer la efectividad de RSI dirigida por la cepa R4 en “Thompson Seedless” con riendo un estado de prealerta.

El objetivo principal fue evaluar el mecanismo de RSI en “Thompson Seedless”, al estímulo de los genes (Lox2, Tlp1, Npr1, Eir1). Se verifico la activación de genes de defensa para hojas y raíces, demostrando la activación sistémica por inoculación de P. veronii R4 en ‘Thompson Seedless’. Mediante q-PCR observamos la expresión del gen Lox2 en hojas de vid, con aumento progresivo hasta (12 h de post inoculación) de muestreo, al analizar este mismo gen en raíces no existió estímulo alguno. Los niveles transcripcionales de los genes Eir1 y Tlp1, fueron estimulados solo en raíces al ser expuestas por R4. Los niveles de estímulo del gen Tlp1 se relaciona con la habilidad que tiene R4 en desarrollar el complejo de simbiosis inducido por la vía del etileno (ET). Los niveles de estímulo del gen Npr1 fueron constitutivos en hojas y raíces, no encontrando diferencias signi cativas entre plantas tratadas con PBS o R4. Los resultados demuestran que R4, en contacto con raíces de ‘Thompson Seedless’, estimulan la expresión de los genes Eir1, Lox2, Tlp1 a 5 min, 6 y 12 h de post inoculación, permitiendo establecer la efectividad de RSI dirigida por la cepa R4 en “Thompson Seedless” con riendo un estado de prealerta.

La photosynthèse oxygénique est bien conservée, des cyanobactéries aux organismes contenant des chloroplastes, tels que les algues et les plantes. Utilisant l'énergie de la lumière, le dioxyde de carbone (CO₂), les minéraux et l'eau, les organismes photosynthétiques assimilent une grande quantité de carbone et d'azote inorganiques pour générer leur biomasse, qui est ensuite distribuée aux organismes hétérotrophes, suggérant qu'ils constituent la base de la chaîne alimentaire. Cependant, ce mécanisme peut produire des espèces réactives de l'oxygène (ROS), qui causent des dommages irréversibles aux protéines à l'appareil photosynthétique, puis réduisant l'efficacité de la photosynthèse. Les organismes photosynthétiques ont donc développé des processus pour diminuer aux ROS générés par la photosynthèse. L'un d'entre eux, appelé acclimatation, permet d'ajuster la composition de l'appareil photosynthétique afin de le rendre tolérant aux conditions fluctuantes. Deux protéines trouvées dans les membranes photosynthétiques à savoir i) d'Acclimatation de la Photosynthèse à l'Environnement 1 (Ape1) et ii) Protéine Luminale Thylakoïdienne 15 (Tlp15), sont conservées et, grâce au criblage génétique, ont été considérées comme impliquées dans le processus d'acclimatation chez la microalgue Chlamydomonas reinhardii et la plante Arabidopsis thaliana. Cependant, le rôle de ces protéines reste inconnu et n'a jamais été étudié chez les cyanobactéries, qui ont développé la photosynthèse oxygénique et sont considérées comme i) l'ancêtre des chloroplastes et ii) cellulaires potentielles pour la production durable de molécules pour la santé et l'énergie. L'objectif de cette thèse est d'analyser le rôle d'Ape1 (Slr0575) et de Tlp15 (Sll1071) chez le modèle cyanobactérien, Synechocystis sp. PCC 6803, qui est génétiquement manipulable et capable de croître en l'absence de photosynthèse grâce au glucose. Tout d'abord, des mutants à délétion des gènes codant ces deux protéines ont été construits en remplaçant par une cassette de résistance aux antibiotiques dans toutes les copies chromosomiques de Synechocystis. Les gènes ape1 et tlp15 sont trouvés dispensables dans les conditions de croissance photoautotrophiques standard. Ensuite, les phénotypes des mutants ont été analysés par des méthodes (croissance dans diverses conditions environnementales et de stress, mesures de l'évolution de l'O₂, tests enzymatiques, quantification des métabolites de certaines molécules carbonées, etc.). Le mutant Δape1 s'est révélé sensible au stress oxydatif (le peroxyde d'hydrogène et la ménadione) et produit plus de ROS que la souche sauvage (WT), suggérant que la protéine Ape1 est impliquée dans la tolérance au stress oxydatif. Le mutant Δtlp15 croît comme la souche WT dans des conditions photoautotrophiques sous diverses intensités lumineuses, mais produit moins d'O₂ que la WT. De manière surprenante, en présence de glucose (conditions mixotrophiques) sous une forte lumière qui favorise le stress oxydatif et la production de méthylglyoxal, le mutant Δtlp15 est capable de croître mais la souche WT ne l'est pas. Ce phénotype est dû à l'absence de tlp15, pas de mutation secondaire, puisque l'introduction du gène tlp15 (complémentation) dans le mutant Δtlp15 rétablit le phénotype WT. Le mutant Δtlp15 s'est également révélé plus tolérant à divers stress oxydatifs et capable de réorganiser les voies du métabolisme du carbone différemment de la souche WT. Une analyse structure-fonction a été réalisée en utilisant la mutagenèse dirigée. Le domaine transmembranaire de Tlp15 et les deux cystéines impliquées dans la formation d'un pont disulfure se sont révélés impliqués dans l'activité de Tlp15. Tous ces résultats suggèrent que Tlp15 participe à un mécanisme de régulation redox impliqué dans le processus d'acclimatation de la photosynthèse. Un modèle intégrant l'ensemble des résultats est proposé
Oxygenic photosynthesis is well-conserved from cyanobacteria to chloroplast-containing organisms such as algae and plants. Using energy from light, carbon dioxide (CO₂), minerals and water, the photosynthetic organisms assimilate a large number of inorganic carbon and nitrogen to generate their biomass to be distributed to the heterotrophic organisms suggesting that they are the base of the food chain. However, the mechanism can lead to the formation of Reactive Oxygen Species (ROS) which causes irreversible damages on proteins, lipids, DNA and photosynthetic apparatus thus decreasing the photosynthesis efficiency. Therefore, photosynthetic organisms had developed various processes to deal with ROS generated directly or indirectly from photosynthesis. Some of them are conserved through the evolution of the green lineage. One of these, named the acclimation, allows adjusting the composition of photosynthetic apparatus in the thylakoid membrane to make them tolerant to fluctuating conditions. Two proteins found in thylakoids, the photosynthetic membranes, i) the Acclimation of Photosynthesis to the Environment 1 protein (Ape1) and the Thylakoid Luminal Protein 15 (TLP15) are conserved through the green lineage and thanks to genetic screening, they were considered to be involved in the acclimation process in the microalga, Chlamydomonas reinhardii, and the plant, Arabidopsis. However, the role of these proteins remained unknown and was never been studied in cyanobacteria that have evolved oxygenic photosynthesis and are considered as i) the ancestor of the chloroplast of eukaryotic photosynthetic organisms, ii) potential cell factories for sustainable production of high added values molecules for health and energy. The objective of this thesis is to analyze the role of Ape1 (Slr0575) and Tlp15 (Sll1071) in the model cyanobacterium, Synechocystis sp. PCC 6803 that is genetically manipulable and is able to grow in the absence of photosynthesis at the expense of glucose. First, single knockout mutants of the genes encoding the two proteins were constructed by replacing their corresponding genes with an antibiotic resistance cassette in all chromosome copies of Synechocystis. ape1 and tlp15 genes were shown to be dispensable in standard photoautotrophic growth. Then the mutant phenotypes were analyzed by a combination of methods (growth in various environmental and stress conditions, O₂ evolution measurements, enzymatic assays, metabolites quantification of some carbonated molecules, etc..). The Δape1 mutant was shown to be sensitive to oxidative stress induced by hydrogen peroxide and menadione and produces more ROS than the wild-type strain (WT) suggesting that APE1 protein is involved in the tolerance to oxidative stress. The Δtlp15 mutant grows as the WT strain in photoautotrophic conditions under various light intensities but produce less O₂ than the WT. Surprisingly, in the presence of glucose (mixotrophic conditions) under high light that promote oxidative stress and methylglyoxal production, the Δtlp15 is able to grow whereas the WT strain is not. This phenotype is due to the absence of tlp15 and not to a secondary mutation since the introduction of tlp15 gene (complementation) in the Δtlp15 restores the WT phenotype. The Δtlp15 mutant was also shown to be more tolerant to various oxidative stresses and is able to reorganize carbon metabolism pathways differently than the WT. A structure function analysis was performed using site-directed mutagenesis. The transmembrane domain of Tlp15 as well as the two cysteines involved in the formation of a disulfide bridge were shown to be involved in the activity of Tlp15. All these results suggest that Tlp15 participates in a redox regulation mechanism involved in photosynthesis acclimation process. A model taking into account all the results is proposed

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 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.