MOLECULAR CLONING, OVERPRODUCTION AND CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS IMP DEHYDROGENASE (EC 1.1.1.205)

Thiago Milech de Assunção (INCT-TB); Cristopher Schneider (INCT-TB); Luiz Augusto Basso (INCT-TB); Diógenes Santiago Santos (INCT-TB)

Mycobacterium tuberculosis, the agent of human tuberculosis (TB), causes about 2 million deaths per year. It is estimated by the World Health Organization that one-third of the world’s population is infected with M. tuberculosis in a latent form. Furthermore, the increasing emergence of multidrug-resistant strains has complicated the treatment and control of TB. Knowledge of the mode of action and role of purine de novo pathway enzymes in M. tuberculosis, which are required for mycobacterial growth, could reveal new targets for the rational design of potent and selective anti-TB agents that might be active against drug-resistant strains. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the nicotinamide-adenine dinucleotide (NAD⁺)-dependent oxidation of inosine 5’-monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). Because this enzyme acts on the synthesis of purine nucleotides at a critical step, it can be essential for mycobacterial growth. The goal of this work is to study and understand the kinetics and biochemistry of the recombinant IMPDH enzyme from M. tuberculosis. The complete DNA coding sequence of IMPDH (EC 1.1.1.205) from M. tuberculosis was obtained using genomic DNA in standard polymerase chain reaction (PCR) conditions. The amplified fragment was cloned into the pCR-Blunt vector and subcloned into the  NdeI/HindIII polylinker region of the pET-23a (+) prokaryotic expression vector. Nucleotide sequence of the cloned fragment was determined by automated DNA sequencing. The recombinant plasmid was then subjected to different expression tests in different Escherichia coli strains. The enzyme was expressed in E. coli BL21 (DE3) cells in the soluble fraction, providing sufficient material for purification by FPLC, biochemistry assays, determination of its three-dimensional structure and study of its key enzymological properties. These results have important implications for understanding the purine metabolism of the TB bacillus and will serve as an initial step toward the future rational design of specific inhibitors and drugs for this M. tuberculosis enzyme.