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 Citations (Scopus)

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 2010
Externally publishedYes

Fingerprint

Cytidine Deaminase
Mycobacterium tuberculosis
Cytidine
Metals
Deoxycytidine
Deoxyuridine
Sequence Alignment
Uridine
Protein Sequence Analysis
Gel Chromatography
Cysteine
Mass Spectrometry
Tuberculosis
Escherichia coli
Enzymes
Pharmaceutical Preparations

Keywords

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

ASJC Scopus subject areas

  • Structural Biology

Cite this

Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase. / Sánchez-Quitian, Zilpa A.; Schneider, Cristopher Z.; Ducati, Rodrigo G.; de Azevedo, Walter F.; Bloch, Carlos; Basso, Luiz A.; Santos, Diógenes S.

In: Journal of Structural Biology, Vol. 169, No. 3, 03.2010, p. 413-423.

Research output: Contribution to journalArticle

Sánchez-Quitian, Zilpa A. ; Schneider, Cristopher Z. ; Ducati, Rodrigo G. ; de Azevedo, Walter F. ; Bloch, Carlos ; Basso, Luiz A. ; Santos, Diógenes S. / Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase. In: Journal of Structural Biology. 2010 ; Vol. 169, No. 3. pp. 413-423.
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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 {\AA} 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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AB - 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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