TY - JOUR
T1 - Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase
AU - Sánchez-Quitian, Zilpa A.
AU - Schneider, Cristopher Z.
AU - Ducati, Rodrigo G.
AU - de Azevedo, Walter F.
AU - Bloch, Carlos
AU - Basso, Luiz A.
AU - Santos, Diógenes S.
N1 - Funding Information:
This work was supported by National Institute of Science and Technology on Tuberculosis (Decit/SCTIE/MS-MCT-CNPq-FNDCT-CAPES) and Millennium Initiative Program (CNPq), Brazil, to D.S.S. and L.A.B., C.B. Jr. acknowledges financial support from “Embrapa Recursos Genéticos e Biotecnologia”, Brazil. D.S.S. (304051/1975-06), L.A.B. (520182/99-5), C.B.J. (304034/2008-8), and W.F.A. Jr. (300851/98-7) are research career awardees of the National Council for Scientific and Technological Development of Brazil (CNPq). This research was partially supported by Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil). Z.A.S.Q. acknowledges a scholarship awarded by CNPq.
PY - 2010/3
Y1 - 2010/3
N2 - 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.
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.
KW - Crystal structure
KW - Cytidine deaminase
KW - Enzyme kinetics
KW - Metalloenzyme
KW - Mycobacterium tuberculosis
KW - Pyrimidine salvage pathway
UR - http://www.scopus.com/inward/record.url?scp=76749150421&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=76749150421&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2009.12.019
DO - 10.1016/j.jsb.2009.12.019
M3 - Article
C2 - 20035876
AN - SCOPUS:76749150421
SN - 1047-8477
VL - 169
SP - 413
EP - 423
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -