Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme

ABSTRACT Cytidine 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 th...

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Autores principales:	Ana Moro-Bulnes, Víctor M. Castillo-Acosta, Maria Valente, Juana Carrero-Lérida, Guiomar Pérez-Moreno, Luis Miguel Ruiz-Pérez, Dolores González-Pacanowska
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Trypanosoma brucei cytidine deaminase pyrimidine metabolism thymidylate biosynthesis Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/8c654b3d67484ac68b293f3a675cf62d
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spelling	oai:doaj.org-article:8c654b3d67484ac68b293f3a675cf62d2021-11-15T15:22:27ZContribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme10.1128/mSphere.00374-192379-5042https://doaj.org/article/8c654b3d67484ac68b293f3a675cf62d2019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00374-19https://doaj.org/toc/2379-5042ABSTRACT Cytidine 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.Ana Moro-BulnesVíctor M. Castillo-AcostaMaria ValenteJuana Carrero-LéridaGuiomar Pérez-MorenoLuis Miguel Ruiz-PérezDolores González-PacanowskaAmerican Society for MicrobiologyarticleTrypanosoma bruceicytidine deaminasepyrimidine metabolismthymidylate biosynthesisMicrobiologyQR1-502ENmSphere, Vol 4, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Trypanosoma brucei cytidine deaminase pyrimidine metabolism thymidylate biosynthesis Microbiology QR1-502
spellingShingle	Trypanosoma brucei cytidine deaminase pyrimidine metabolism thymidylate biosynthesis Microbiology QR1-502 Ana Moro-Bulnes Víctor M. Castillo-Acosta Maria Valente Juana Carrero-Lérida Guiomar Pérez-Moreno Luis Miguel Ruiz-Pérez Dolores González-Pacanowska Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme
description	ABSTRACT Cytidine 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.
format	article
author	Ana Moro-Bulnes Víctor M. Castillo-Acosta Maria Valente Juana Carrero-Lérida Guiomar Pérez-Moreno Luis Miguel Ruiz-Pérez Dolores González-Pacanowska
author_facet	Ana Moro-Bulnes Víctor M. Castillo-Acosta Maria Valente Juana Carrero-Lérida Guiomar Pérez-Moreno Luis Miguel Ruiz-Pérez Dolores González-Pacanowska
author_sort	Ana Moro-Bulnes
title	Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme
title_short	Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme
title_full	Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme
title_fullStr	Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme
title_full_unstemmed	Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme
title_sort	contribution of cytidine deaminase to thymidylate biosynthesis in <named-content content-type="genus-species">trypanosoma brucei</named-content>: intracellular localization and properties of the enzyme
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/8c654b3d67484ac68b293f3a675cf62d
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Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in <named-content content-type="genus-species">Trypanosoma brucei</named-content>: Intracellular Localization and Properties of the Enzyme

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