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

Zilpa A. Sánchez-Quitian, Cristopher Z. Schneider, Rodrigo G. Ducati, Walter F. de Azevedo, Carlos Bloch, Luiz A. Basso, Diógenes S. Santos

Research output: Contribution to journalArticle

13 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)413-423
Number of pages11
JournalJournal of Structural Biology
Volume169
Issue number3
DOIs
StatePublished - Mar 1 2010

Keywords

  • Crystal structure
  • Cytidine deaminase
  • Enzyme kinetics
  • Metalloenzyme
  • Mycobacterium tuberculosis
  • Pyrimidine salvage pathway

ASJC Scopus subject areas

  • Structural Biology

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    Sánchez-Quitian, Z. A., Schneider, C. Z., Ducati, R. G., de Azevedo, W. F., Bloch, C., Basso, L. A., & Santos, D. S. (2010). Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase. Journal of Structural Biology, 169(3), 413-423. https://doi.org/10.1016/j.jsb.2009.12.019