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 microM and k(cat)=4.8s(-1) for cytidine, and K(m)=1059 microM and k(cat)=3.5s(-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 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.

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

“…8,9 The proton and the hydroxyl anion add to, respectively, the N3 and the C4 atoms of the N3]C4 double bond of cytidine to form a tetrahedral intermediate in the rst step. [18][19][20][21] In previous pH-rate prole studies of MtCDA, protonation of a single ionizable group with an apparent pK a value of 4.3 (AE1) reduced the catalytic activity and protonation of a group with apparent pK a of 4.7 (AE0.7) appeared to be involved in cytidine binding. 8,9 Hydrogen bonding plays an important role in transition state stabilization for E. coli CDA, and the carboxymethyl group of Glu104 appears to minimize the activation barrier for deamination, not only by stabilizing the altered substrate in the transition state but also by destabilizing the enzyme-substrate and enzyme-product complexes.…”

“…On the other hand, the pyrimidine salvage pathway reutilize nucleosides and pyrimidine bases derived from preformed nucleotides being preferentially utilized by bacteria under restricted energy availability, because it demands less energy than the de novo biosynthesis. [17][18][19] D-CDA consists of two symmetrical subunits, each monomer containing three domains: a small N-terminal domain, a catalytic domain containing a zinc atom, and a Cterminal domain, that contains a cavity described as "broken active site". 6 Accordingly, the pyrimidine salvage pathway enzymes are interesting targets due to low energy demand to synthesize nucleotides.…”

“…Cytidine deaminase enzyme (CDA, cytidine/2 0 -deoxycytidine aminohydrolase; EC 3.5.4.5) catalyzes the deamination of cytidine/2 0 -deoxycytidine to form uridine/2 0 -deoxyuridine. 19 Each monomer has ve b-strands and ve a-helices. 8,9 The proton and the hydroxyl anion add to, respectively, the N3 and the C4 atoms of the N3]C4 double bond of cytidine to form a tetrahedral intermediate in the rst step.…”

“…7 The proposed mechanism of catalysis for Escherichia coli CDA includes a zinc atom in the active site that activates a water molecule to form a hydroxide ion and H + by heterolytic bond cleavage. 20 The human CDA region involved in substrate binding contains the residues 32 18,19,21 Conserved residues in tetrameric CDA enzyme involved in catalysis are 65 CAERTA 70 for human CDA, 53 CAERTA 58 for B. subtilis CDA, and 56 CAECAV 61 for MtCDA. In the second step, uridine is produced with the release of ammonia.…”

Metallobiology of Tuberculosis

“…The number of Zn containing proteins identified in mycobacteria has increased significantly as more protein structures are resolved. Zn is part of M. tuberculosis zinc-metallopeptidases (33, 34), carbonic anhydrase (35), fructose biphosphate aldolase Fba (36), the helicase RqlH (37), the cytidine deaminase Cda (38), the MshC ligase involved in mycothiol biosynthesis (39), the 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase IspF (40), the 2-isopropylmalate synthase LeuA involved in leucine biosynthesis (41), the superoxide dismutase (SOD) SodC (42, 43), the Esx-3 substrate EsxG-EsxH complex (44), the inositol 1-phosphate synthase (45), the RecA intein (46) and several more.…”