Cloning, Expression And Characterization Of The Gene Encoding Enoyl-CoA Hydratase In Mycobacterium Tuberculosis

Several enzymes of the fatty acid biosynthesis pathway are targeted by anti-tuberculosis drugs such as isoniazid for the treatment of tuberculosis; mutations arising in enzymes of this pathway have.been shown to confer drug resistance to a variety of Myco-bacterium tuberculosis strains, leading to the resurgence of this disease in an alarming way in the world.The enoyl-CoA hydratase (echA, EC 4.2.1.17) is responsible for me-tabolization of mycolic acid in Mycobacterium tuberculosis in vivo, which is a kind of bioactive key enzyme in cell wall and involved in a number of biochemical reactions. M. tuberculosis genome has a group of 21 enzymes belonging to enoyl-CoA hydratase/iso-merase superfamily and been predicted that these enzymes are synthesized as intrace-llular proteins. Some enoyl-CoA hydratases are not functionally linked, whereas each gene product is functionally linked with other echA family members, thus these enzymes may have distinct substrate specificities.In this study, the coding sequence for Mycobacterium tuberculosis (H37Rv) echA18 was cloned into the prokaryotic expression vector pET32a(+) and expressed in Esche-richia coli BL21(DE3). A major band corresponding to a protein of 42kDa was detected on SDS-PAGE. The protein was distributed in both the soluble fraction and the insoluble fraction. In soluble fraction the protein was purificated. The secondary structure feature of the protein was determined by circular dichroism (CD) and its possible three- dimensional model was constructed by molecular modeling technique. The amino acid sequence analysis showed that the M.tuberculosis echA18 is similar to rat enoyl-CoA hydratase which indicated 39% identity in amino acid sequences. The transmembrane (TM) segment of echA18 protein is relatively more conserved in the whole protein than other region, while the C-terminal homology is the poorest. These conserved sites may be essentially associated with the function of echA18. Based on the secondary structure prediction, a primary 3D structure model of echA18 protein was constructed by molecular modeling method.