Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization

Autores
Lobo Rojas, Ángel E.; González Marcano, Eglys B.; Valera Vera, Edward Augusto; Acosta, Rodolfo Héctor; Quiñones, Wilfredo A; Burchmore, Richard J. S.; Concepción, Juan L.
Año de publicación
2016
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9 kDa and 51.3 kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108 mM (TcGALK-1) and 0.091 mM (TcGALK-2) and for ATP 0.36 mM (TcGALK-1) and 0.1 mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414 mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52-47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26-23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20 mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.
Fil: Lobo Rojas, Ángel E.. Universidad de los Andes; Venezuela
Fil: González Marcano, Eglys B.. Universidad de los Andes; Venezuela
Fil: Valera Vera, Edward Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Universidad de los Andes; Venezuela
Fil: Acosta, Rodolfo Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de los Andes; Venezuela
Fil: Quiñones, Wilfredo A. Universidad de los Andes; Venezuela
Fil: Burchmore, Richard J. S.. University of Glasgow; Reino Unido
Fil: Concepción, Juan L.. Universidad de los Andes; Venezuela
Materia
Galactokinase
Galactose Metabolism
Ghmp Superfamily
Glycosome
Kinetic Analysis
Nucleotide Sugars
Trypanosoma Cruzi
Udp-Galactose
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/83377
Acceder
id CONICETDig_cbc615b4f860e573e3ecd915bf7c40fb
oai_identifier_str oai:ri.conicet.gov.ar:11336/83377
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterizationLobo Rojas, Ángel E.González Marcano, Eglys B.Valera Vera, Edward AugustoAcosta, Rodolfo HéctorQuiñones, Wilfredo ABurchmore, Richard J. S.Concepción, Juan L.GalactokinaseGalactose MetabolismGhmp SuperfamilyGlycosomeKinetic AnalysisNucleotide SugarsTrypanosoma CruziUdp-Galactosehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9 kDa and 51.3 kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108 mM (TcGALK-1) and 0.091 mM (TcGALK-2) and for ATP 0.36 mM (TcGALK-1) and 0.1 mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414 mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52-47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26-23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20 mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.Fil: Lobo Rojas, Ángel E.. Universidad de los Andes; VenezuelaFil: González Marcano, Eglys B.. Universidad de los Andes; VenezuelaFil: Valera Vera, Edward Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Universidad de los Andes; VenezuelaFil: Acosta, Rodolfo Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de los Andes; VenezuelaFil: Quiñones, Wilfredo A. Universidad de los Andes; VenezuelaFil: Burchmore, Richard J. S.. University of Glasgow; Reino UnidoFil: Concepción, Juan L.. Universidad de los Andes; VenezuelaElsevier Ireland2016-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/83377Lobo Rojas, Ángel E.; González Marcano, Eglys B.; Valera Vera, Edward Augusto; Acosta, Rodolfo Héctor; Quiñones, Wilfredo A; et al.; Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization; Elsevier Ireland; Parasitology International; 65; 5; 10-2016; 472-4821383-5769CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S1383576916301891info:eu-repo/semantics/altIdentifier/doi/10.1016/j.parint.2016.06.008info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:52:45Zoai:ri.conicet.gov.ar:11336/83377instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:52:46.108CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
title Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
spellingShingle Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
Lobo Rojas, Ángel E.
Galactokinase
Galactose Metabolism
Ghmp Superfamily
Glycosome
Kinetic Analysis
Nucleotide Sugars
Trypanosoma Cruzi
Udp-Galactose
title_short Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
title_full Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
title_fullStr Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
title_full_unstemmed Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
title_sort Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization
dc.creator.none.fl_str_mv Lobo Rojas, Ángel E.
González Marcano, Eglys B.
Valera Vera, Edward Augusto
Acosta, Rodolfo Héctor
Quiñones, Wilfredo A
Burchmore, Richard J. S.
Concepción, Juan L.
author Lobo Rojas, Ángel E.
author_facet Lobo Rojas, Ángel E.
González Marcano, Eglys B.
Valera Vera, Edward Augusto
Acosta, Rodolfo Héctor
Quiñones, Wilfredo A
Burchmore, Richard J. S.
Concepción, Juan L.
author_role author
author2 González Marcano, Eglys B.
Valera Vera, Edward Augusto
Acosta, Rodolfo Héctor
Quiñones, Wilfredo A
Burchmore, Richard J. S.
Concepción, Juan L.
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Galactokinase
Galactose Metabolism
Ghmp Superfamily
Glycosome
Kinetic Analysis
Nucleotide Sugars
Trypanosoma Cruzi
Udp-Galactose
topic Galactokinase
Galactose Metabolism
Ghmp Superfamily
Glycosome
Kinetic Analysis
Nucleotide Sugars
Trypanosoma Cruzi
Udp-Galactose
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9 kDa and 51.3 kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108 mM (TcGALK-1) and 0.091 mM (TcGALK-2) and for ATP 0.36 mM (TcGALK-1) and 0.1 mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414 mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52-47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26-23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20 mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.
Fil: Lobo Rojas, Ángel E.. Universidad de los Andes; Venezuela
Fil: González Marcano, Eglys B.. Universidad de los Andes; Venezuela
Fil: Valera Vera, Edward Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Universidad de los Andes; Venezuela
Fil: Acosta, Rodolfo Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de los Andes; Venezuela
Fil: Quiñones, Wilfredo A. Universidad de los Andes; Venezuela
Fil: Burchmore, Richard J. S.. University of Glasgow; Reino Unido
Fil: Concepción, Juan L.. Universidad de los Andes; Venezuela
description Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9 kDa and 51.3 kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108 mM (TcGALK-1) and 0.091 mM (TcGALK-2) and for ATP 0.36 mM (TcGALK-1) and 0.1 mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414 mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52-47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26-23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20 mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.
publishDate 2016
dc.date.none.fl_str_mv 2016-10
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/83377
Lobo Rojas, Ángel E.; González Marcano, Eglys B.; Valera Vera, Edward Augusto; Acosta, Rodolfo Héctor; Quiñones, Wilfredo A; et al.; Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization; Elsevier Ireland; Parasitology International; 65; 5; 10-2016; 472-482
1383-5769
CONICET Digital
CONICET
url http://hdl.handle.net/11336/83377
identifier_str_mv Lobo Rojas, Ángel E.; González Marcano, Eglys B.; Valera Vera, Edward Augusto; Acosta, Rodolfo Héctor; Quiñones, Wilfredo A; et al.; Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization; Elsevier Ireland; Parasitology International; 65; 5; 10-2016; 472-482
1383-5769
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S1383576916301891
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.parint.2016.06.008
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Ireland
publisher.none.fl_str_mv Elsevier Ireland
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.13397

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