Mycobacterium Tuberculosis Fatty Acid Metabolic Pathways, Enzymes And Protein Molecular Biology And Three-dimensional Structure Of The Study

Tuberculosis is one of the world's most devastating diseases, with more than three million deaths and eight million new cases occurring annually. Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Despite the availability of effective short-course chemotherapy (DOTS) and the Bacille Calmette-Gue'rin (BCG) vaccine, M. tuberculosis continues to claim more lives than any other single infectious agent. The increasing emergence of drug-resistant tuberculosis along with the HIV pandemic threatens disease control and highlights both the need to understand how our current drugs work and the need to develop new and more effective drugs.Like many organisms, the whole genome of M. tuberculosis has now been entirely sequenced. The analysis of this bacterium's genome sequence has itself led to informative hypotheses on its biology. The original sequence and annotation of M tuberculosis strain H37Rv identified 4056 genes, which included 4006 genes thought to encode proteins and 50 encoding stable RNA. The cell wall of Mycobacterium tuberculosis is based on a conventional peptidoglycan, which covalently linked to an arabinogalactan esterified with mycolic acids. Mycolic acids, which are the largest fatty acids in nature, form the basis of a complicated lipid bilayer outer membrane, and represent 40-60% by weight of the mycobacterial cell envelope. Malonyl coenzyme A (CoA)-ACP transacylase (MCAT) is a key enzyme in the FAS II pathway of bacteria. MCAT catalyzes the transfer of a malonyl moiety from malonyl-CoA to holo-ACP, generating free CoASH and malonyl-ACP. Acyl carrier protein (ACP) is a small, acidic protein in FAS II systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Malonyl-ACP, a substrate for the elongation steps in the fatty acid biosynthesis, indicated that MCATs and ACPs maybe the building block of the cycle.In our study, Rv0033, Rv0069, Rv1344, Rv2243, Rv2244 were inserted into a bacterial expression vector pET28a resulting in the 6-Histidine-tag recombinant plasmids. Proteins were purified by nickel affinity chromatography and their characterizations have been investigated. The molecular weights of proteins were estimated by MALDI-TOF. MCAT genes RT-PCR results reveal different transcript condition with each other. In order to study the role of the conserved residues and the conformation of Acp1344, some site-directed mutations of recombinant protein were made and the 3D structure of Acpl344 was modeled. For providing the structure bases on drug design, the MCAT proteins were expressed in E.coli and puried using GST or Ni²⁺-NTA affinity chromatography, gel filtration chromatography and Ion-exchange chromatography. The crystals were obtained by using hanging-drop vapour-diffusion method in a crystallization condition of MCAT.