Functional and structural evidence for the catalytic role played by glutamate-47 residue in the mode of action of Mycobacterium tuberculosis cytidine deaminase

Abstract

Strategies to combat tuberculosis (TB) are needed to kill drug-resistant strains and be effective against latent Mycobacterium tuberculosis, the causative agent of TB. Cytidine deaminase (CDA) catalyzes the hydrolytic deamination of cytidine to uridine, and belongs to the pyrimidine salvage pathway. The CDA from M. tuberculosis (MtCDA) is a target for the development of drugs against TB because it may be involved in latency mechanisms. The role of the conserved glutamate-47 (E47) residue was evaluated by construction of five mutant proteins (E47A, E47D, E47L, E47H, and E47Q). Mutants E47A and E47H were expressed in the insoluble fraction, whereas E47D, E47L and E47Q were soluble and purified. The E47D, E47L and E47Q mutants contained 1 mol of Zn²⁺ per mol of protein subunit. These mutations had no effect on the oligomerization state of MtCDA. Steady-state kinetic results showed that K_M values for the E47D and E47Q mutants were not significantly altered, whereas there was a decrease in k_cat values of 37-fold for E47D and 19-fold for E47Q mutant. No activity could be detected for E47L mutant. No k_cat and k_cat/K_M dependence on pH values ranging from 4.0 to 11 were observed for E47D mutant from pH-rate profiles. A catalytic role was proposed for the γ-carboxyl group of E47, and its likely involvement in the stabilization of the transition state was suggested. Structural comparisons between E47D and E47Q mutants with the apo and holo forms of wild-type MtCDA reveal subtle differences that support this proposal.