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: K-m = 1004 mu M and k(cat) = 4.8 s(-1) for cytidine, and K-m = 1059 mu M 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 pK(a) value of 4.3 abolishes activity, and protonation of a group with pK(a) value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 angstrom 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. (C) 2009 Elsevier Inc. All rights reserved.