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dc.contributor.authorMoro-Bulnes, Ana-
dc.contributor.authorCastillo-Acosta, Victor M.-
dc.contributor.authorValente, María-
dc.contributor.authorCarrero-Lérida, Juana-
dc.contributor.authorPérez-Moreno, Guiomar-
dc.contributor.authorRuiz-Pérez, Luis Miguel-
dc.contributor.authorGonzález-Pacanowska, D.-
dc.identifierdoi: 10.1128/mSphere.00374-19-
dc.identifierissn: 2379-5042-
dc.identifier.citationmSphere 4 (2019)-
dc.description.abstractCytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of Trypanosoma brucei CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2′-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs T. brucei proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for de novo thymidylate biosynthesis. This observation contrasts with the existence in T. brucei of a dimeric deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, T. brucei dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in T. brucei, CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism. IMPORTANCE Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in Trypanosoma brucei exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant T. brucei CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5′-triphosphate nucleotidohydrolase does not have a prominent role in de novo dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway-
dc.description.sponsorshipWe thank Aurora Constan for technical assistance and Santiago Castanys for critical reading of the manuscript. We thank the TrypTag database for the available information regarding the subcellular localization of TMPK and C1-THF synthase. This work was funded by the Junta de Andalucía (BIO-199 P12-BIO-2059), the Plan Nacional de Investigación Científica, Instituto de Salud Carlos III—Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014), the Plan Nacional (SAF2016-79957- R), and the FEDER funds from the EU. A.M.-B. was supported by a Formación del Profesorado Universitario (Ref FPU15/01820) fellowship from the Spanish Government (Ministerio de Educación y Formación Profesional). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.-
dc.publisherAmerican Society for Microbiology-
dc.relation.isversionofPublisher's version-
dc.subjectTrypanosoma brucei, cytidine deaminase, pyrimidine metabolism, thymidylate biosynthesis-
dc.titleContribution of Cytidine Deaminase to Thymidylate Biosynthesis in Trypanosoma brucei: Intracellular Localization and Properties of the Enzyme-
dc.contributor.funderJunta de Andalucía-
dc.contributor.funderInstituto de Salud Carlos III-
dc.contributor.funderRed de Investigación Cooperativa en Enfermedades Tropicales (España)-
dc.contributor.funderMinisterio de Economía y Competitividad (España)-
dc.contributor.funderMinisterio de Educación y Formación Profesional (España)-
dc.contributor.funderEuropean Commission-
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