URACIL PHOSPHORIBOSYLTRANSFERASE FROM MYCOBACTERIUM TUBERCULOSIS H37RV AS A TARGET FOR ANTI-TB RATIONAL DRUG DESIGN AND VACCINE DEVELOPMENT

Anne Drumond Villela (CPBMF INCT-TB PUCRS); Cristopher Zandoná Schneider (CPBMF INCT-TB PUCRS); Luiz Augusto Basso (CPBMF INCT-TB PUCRS); Diógenes Santiago Santos (CPBMF INCT-TB PUCRS)

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. The World Health Organization estimates that 9.27 million new TB cases occurred in 2007, resulting in 2 million deaths worldwide. There is a critical need for the development of vaccines to prevent initial infection and of drugs that would reduce progression from latent M. tuberculosis infection to active TB. The gene (upp, Rv3309c) encoding uracil phosphoribosyltransferase (UPRTase) was identified by homology in the genome of M. tuberculosis H37Rv. The UPRTase (EC 2.4.2.9) enzyme from M. tuberculosis is part of the pyrimidine salvage pathway and catalyzes the conversion of uracil and 5-phosphate-a-1-diphosphate (PRPP) to uridine 5'-monophosphate (UMP) and diphosphate, respectively. UPRTase from M. tuberculosis is an attractive target for rational drug design against TB, since it may have an important role in the latent stage of the TB bacillus. The goals of this work include amplification, cloning, overexpression, purification and characterization of recombinant UPRTase from M. tuberculosis to validate its biological role as an UPRTase and its relevancy for the pyrimidine salvage pathway in the M. tuberculosis metabolism. The upp gene (624 bp) was amplified from M. tuberculosis H37Rv genomic DNA. Then, it was cloned into the pCR-Blunt vector and subcloned into the pET-23a(+) expression vector, which was used to express the recombinant protein in BL21(DE3) Escherichia coli cells. Three chromatography steps were employed to obtain homogeneous recombinant M. tuberculosis UPRTase. Mass spectrometry analysis and N-terminal amino acid sequencing provided evidence for the identity of the enzyme. M. tuberculosis UPRTase activity was determined spectrophotometrically by measuring the conversion of uracil into UMP at 280 nm. Steady-state kinetic analyses will provide evidence for the kinetic mechanism of this enzyme, which may be useful for the rational design of future anti-TB agents. Moreover, a M. tuberculosis mutant strain for the upp gene is under construction to evaluate the biological role of UPRTase in M. tuberculosis metabolism. This mutant strain might prove to be useful for future development of a new TB vaccine.