title: 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.
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga, 6681, Porto Alegre, RS 90619-900, Brazil.
authors.forename: Zilpa A
authors.initials: ZA
authors.lastname: Sánchez-Quitian
authors.forename: Cristopher Z
authors.initials: CZ
authors.lastname: Schneider
authors.forename: Rodrigo G
authors.initials: RG
authors.lastname: Ducati
authors.forename: Walter F
authors.initials: WF
authors.lastname: de Azevedo
authors.forename: Carlos
authors.initials: C
authors.lastname: Bloch
authors.forename: Luiz A
authors.initials: LA
authors.lastname: Basso
authors.forename: Diógenes S
authors.initials: DS
authors.lastname: Santos
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003564:Cytidine Deaminase; D015032:Zinc