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2012. Genre/Form: Academic theses Material Type: Thesis/dissertation, Manuscript, Internet resource Document Type: Book, Archival Material, Internet Resource All Authors / Contributors: Ehab N M Jirgis Find more information about: Ehab N M Jirgis OCLC Number: 812820319 Notes: A thesis submitted in partial fulfilment of the requirement for the degree of Master of Science in Biological Science, The University of Auckland, July 2012. Description: xiv, 91 leaves : illustrations (some color) ; 30 cm Responsibility: Ehab N.M Jirgis. Abstract: Mycobacterium tuberculosis (Mtb) is a Gram-positive pathogenic bacterium which is the causative agent of pulmonary tuberculosis (TB). It is estimated that Mtb infects one-third of the world's population, while TB claims approximately two million lives a year. TB can persist in a host for decades and relapse following immune-compromisation. The persistence of TB has been attributed to cellular processes involved in hypoxic metabolism. Menaquinone is an electron carrier important for electron transport across the membrane and energy production during aerobic and anaerobic respiration. The aim of this thesis was express three essential Mtb enzymes involved in menaquinone biosynthesis, MenD, MenC and MenE, with the aim of solving the structure of one of them. These enzymes, along with MenH, catalyse the conversion of isochorismate through to o-Succinylbenzoate-CoA followed by a series of subsequent enzymatic steps to produce menaquinone. MenD and MenC were both expressed in the soluble form using the M. smegmatis expression system. MenD was successfully crystallized in the orthorhombic system with space group P212121, and its crystal structure, both in its apo form and in complex with its cofactor thiamine diphosphate (ThDP), was solved using MR with the BsMenD structure. Four monomers were observed per asymmetric unit forming a dimer of dimers, each monomer comprising three domains typical of ThDP dependent enzymes. Dimer formation was found to be mediated by the interaction of domain I and III of two monomers. Two ThDP binding sites are formed following dimerization and ThDP was found to bind to the protein through interactions with 9 conserved residues. Eight residues from domain III bind to the diphosphate moiety of ThDP, locking it within the protein. Hydrophobic residues from domain III help coordinate the pyrimidine ring for the vital deprotonating interaction with Glu55 from domain I and subsequent activation of catalysis of MtbMenD. Attempts to solve the crystal structure with the isochorismate substrate in complex with MtbMenD were not successful.