Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a pathogenic bacterium that currently infects one-third of the world's population. Although there are effective drugs for TB treatment, the disease is not properly controlled in many poor and developing countries. Drugs that can act against strains of M. tuberculosis would contribute to effective control of this disease. A recent approach for developing new anti-TB agents is rational drug design. Therefore, studies of possible new molecular microbial targets are fundamental.
Enzymes of purine biosynthesis in M. tuberculosis, which are required for mycobacterial growth, have been extensively studied by our research group, providing many novel targets for rational design of more effective antimycobacterial agents.
Adenylosuccinate synthase governs the committed step in the de novo biosynthesis of AMP, coupling the hydrolysis of GTP with the synthesis of adenylosuccinate from L-aspartate and IMP. Because this enzyme acts on the synthesis of purine nucleotides at a critical step, it can be essential for mycobacterial growth. Accordingly, structural and functional studies should be undertaken in order to explore differences in the mode of action and relevant features for this M. tuberculosis enzyme.
DNA sequence of Adenylosuccinate Synthase (EC 6.3.4.4) from M. tuberculosis H37Rv was obtained from public databases. The corresponding purA coding sequence was amplified from genomic DNA using specific primers and PCR standard conditions. The obtained fragment was cloned into the pCR-Blunt vector (Invitrogen) and subcloned into the pET-23a(+) expression vector (Novagen). Nucleotide sequence of the cloned fragment was determined by automated DNA sequencing. Different strains of Escherichia coli were tested for expression of the recombinant enzyme. The enzyme was expressed in E. coli BL21 (DE3) cells in the insoluble fraction, and solubilization was achieved using a freeze-thaw protocol. Optimization of protein expression in other E. coli strains and enzyme solubilization protocols are currently being performed to provide material enough for purification of the recombinant protein. Further characterization of the enzyme three-dimensional structure and study of its enzymological properties will be determined later.