Add time:08/03/2019         Source:sciencedirect.com

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2′-deoxycytidine for uridine and 2′-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn2+-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: Km = 1004 μM and kcat = 4.8 s−1 for cytidine, and Km = 1059 μM and kcat = 3.5 s−1 for 2′-deoxycytidine. The pH dependence of kcat and kcat/KM for cytidine indicate that protonation of a single ionizable group with apparent pKa value of 4.3 abolishes activity, and protonation of a group with pKa value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 Å resolution. Analysis of the crystallographic structure indicated the presence of a Zn2+ coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.

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