STUDIES OF UMP KINASE (EC 2.7.4.-) FROM MYCOBACTERIUM TUBERCULOSIS AS A TARGET FOR PROTEIN INHIBITION

Diana Carolina Rostirolla (INCTT); Ardala Breda (INCTT); Luiz Augusto Basso (INCTT); Diógenes Santiago Santos (INCTT)

Millions of people have died from tuberculosis (TB), a chronic infectious disease mainly caused by Mycobacterium tuberculosis. Despite the availability of effective chemotherapy and the moderately protective vaccine, the tubercle bacillus continues to claim more lives than any other single infectious agent and led us to focus on the development of new drugs to combat the disease. Nucleotide biosynthesis is an essential step in the progression of TB, therefore UMP kinase, a protein of the pyrimidine metabolism and encoded by the pyrH gene, which catalyses the phosphorylation of UMP to UDP, is an attractive antitubercular drug target. The full-length pyrH coding region (786bp) was PCR amplified from M. tuberculosis H37Rv genomic DNA. The PCR fragment was cloned at the NdeI and HindIII restriction sites of the pET-23a(+) expression vector and the resulting plasmid was transformed into the E. coli BL21(DE3) strain and the colonies were grown in TB medium at 30°C and harvested by centrifugation after 24h without induction of IPTG. The recombinant UMP kinase was purified by a three-step protocol using a FPLC system and the activity was measured spectrophotometrically. The oligomeric state of UMP kinase was determined by size exclusion liquid chromatography which suggested that UMP kinase is a tetramer in solution. The molecular mass of the M. tuberculosis UMP kinase subunit (27 264.08 Da) was determined by ESI-MS analysis and protein identity was corroborated by N-termini sequencing. This work represents the first characterization of the recombinant UMP kinase from M. tuberculosis H37Rv overexpressed in a prokaryotic system. These results will allow more detailed studies about the catalytic properties of the enzyme as its substrate specificity and determination of kinetic constants, which represent an essential step in our efforts to characterize a target for anti-tuberculosis drug development.