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.