Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase.

Abstract 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 Zn 2+ -dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K m  = 1004 μM and k cat  = 4.8 s −1 for cytidine, and K m  = 1059 μM and k cat  = 3.5 s −1 for 2′-deoxycytidine. The pH dependence of k cat and k cat / K M for cytidine indicate that protonation of a single ionizable group with apparent p K a value of 4.3 abolishes activity, and protonation of a group with p K a value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 A resolution. Analysis of the crystallographic structure indicated the presence of a Zn 2+ coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.