BIOCHEMICAL AND STRUCTURAL STUDIES OF HISTIDINOL DEHYDROGENASE FROM MYCOBACTERIUM TUBERCULOSIS

José Eduardo Sacconi Nunes (INCT-TB); Rodrigo Gay Ducati (INCT-TB); Ardala Breda (INCT-TB); Bibiana Monson de Souza (INCT-TB); Mario Sergio Palma (INCT-TB); Luiz Augusto Basso (INCT-TB); Diógenes Santiago Santos (INCT-TB)

The aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis, was responsible for approximately 1.8 million deaths in the year of 2007. The increasing prevalence of TB, the emergence of multidrug-resistant and extensively drug-resistant strains of the tubercle bacillus, and the devastating effect of co-infection with the human immunodeficiency virus have led to an urgent need for the development of new and more efficient antimycobacterial drugs. The histidine biosynthetic pathway is present in bacteria, archaebacteria, lower eukaryotes and plants, but is absent in mammals. Disruption of the M. tuberculosis hisD-encoded L-histidinol dehydrogenase gene by homologous recombination resulted in mutants that were not viable under histidine deprivation. Thus, the gene products of this pathway represent attractive targets for the development of antimycobacterial agents. Although analysis of the complete genome sequence of M. tuberculosis predicts that the locus-tag Rv1599 (hisD) codes for a probable histidinol dehydrogenase (MtHisD) enzyme, experimental evidence was lacking. The M. tuberculosis hisD gene was PCR amplified, cloned, sequenced, and the recombinant protein was overexpressed and purified. MtHisD enzyme activity measurements of soluble protein cellular extracts of recombinant protein demonstrated a 612-fold increase in specific activity as compared to control cell extracts. Furthermore, steady-state velocity of recombinant MtHisD showed a linear dependence on cell extract volume added to the reaction mixture. Apparent steady-state kinetic constants were determined with the pure enzyme. The results reported here validate hisD as the structural gene that codes for histidinol dehydrogenase in M. tuberculosis, and along with its proposed three-dimensional model, provide material for structural and mechanistic studies aiming at the rational design of new anti-TB agents.