THE CYTIDINE DEAMINASE FROM MYCOBACTERIUM TUBERCULOSIS H37RV AS A TARGET FOR THE DEVELOPMENT OF NEW ANTI-TUBERCULAR DRUGS

Jacqueline Gonçalves Rehm (INCTT); Zilpa Adriana Quitian Sánchez (INCTT); Rodrigo Gay Ducati (INCTT); Rafael Andrade Caceres (INCTT); Walter Filgueira de Azevedo Jr (INCTT); Luiz Augusto Basso (INCTT); Diógenes Santiago Santos (INCTT)

Human tuberculosis (TB), mainly caused by Mycobacterium tuberculosis, remains a leading cause of mortality worldwide. The emergence of drug-resistant strains has exacerbated the treatment and control of TB. There is thus a continuous need to identify promising targets for the development of anti-TB agents. Cytidine deaminase (CDA) is an attractive target for drug development because it is a pyrimidine salvage pathway enzyme that recycles cytidine and 2’-deoxycytidine to uridine and 2’-deoxyuridine synthesis, respectively. The objectives of this work were obtain homogeneous CDA from M. tuberculosis in sufficient quantities for structural studies of this enzyme in complex with its substrates and products in order to elucidate its tridimensional structure and catalytic mechanism. The CDA gene from M. tuberculosis H37Rv was amplified, cloned, and sequenced, and the respective recombinant enzyme overexpressed in Escherichia coli BL21(DE3) host cells in the active form. Recombinant CDA was purified to homogeneity through three chromatographic steps with the following columns: Q-Sepharose Fast Flow, Sephacryl-S200, and Butyl Sepharose Fast Flow. The purified protein was concentrated to 12 mg mL^-1 against 20 mM TrisHCl pH 7.5 and incubated overnight with 5 mM of each ligand (cytidine, uridine, 2’-deoxycytidine, and 2’-deoxyuridine). Hanging drops were equilibrated by vapor diffusion at 18°C against reservoir solution containing 0.1 M HEPES pH 7.5 with Sodium chloride 4.3 M. Crystals were formed after 24 hours and the X-ray diffraction data were collected using the synchrotron radiation source (Station PCr, Laboratório Nacional de Luz Síncrontron, LNLS, Campinas, Brazil) and a CCD detector (MARCCD). This work will provide an improved understanding about the enzyme catalytic and chemical mechanism and these data may help in the rational design of inhibitors of CDA from M. tuberculosis, aiming at the development of anti-TB agents.