A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

Autores
Asención Diez, Matías Damián; Ebrecht, Ana Cristina; Martínez, Lucila Inés; Aleanzi, Mabel Cristina; Guerrero, Sergio Adrian; Ballicora, Miguel A.; Iglesias, Alberto Alvaro
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans . In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.
Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Ballicora, Miguel A.. University Of Chicago; Estados Unidos de América;
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Materia
protein engineering
allosteric regulation
pyrophosphorylases evolution
UDP-glucose
ADP-glucose
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/1621
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/1621
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric RegulatorsAsención Diez, Matías DamiánEbrecht, Ana CristinaMartínez, Lucila InésAleanzi, Mabel CristinaGuerrero, Sergio AdrianBallicora, Miguel A.Iglesias, Alberto Alvaroprotein engineeringallosteric regulationpyrophosphorylases evolutionUDP-glucoseADP-glucosehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans . In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Ballicora, Miguel A.. University Of Chicago; Estados Unidos de América;Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Molecular Diversity Preservation International2013-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/1621Asención Diez, Matías Damián; Ebrecht, Ana Cristina; Martínez, Lucila Inés; Aleanzi, Mabel Cristina; Guerrero, Sergio Adrian; et al.; A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators; Molecular Diversity Preservation International; International Journal Of Molecular Sciences; 14; 5; 5-2013; 9703-97211422-0067enginfo:eu-repo/semantics/altIdentifier/doi/10.3390/ijms14059703info:eu-repo/semantics/altIdentifier/url/http://www.mdpi.com/1422-0067/14/5/9703info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T14:22:37Zoai:ri.conicet.gov.ar:11336/1621instacron: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:34982025-10-15 14:22:37.391CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
title A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
spellingShingle A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
Asención Diez, Matías Damián
protein engineering
allosteric regulation
pyrophosphorylases evolution
UDP-glucose
ADP-glucose
title_short A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
title_full A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
title_fullStr A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
title_full_unstemmed A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
title_sort A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators
dc.creator.none.fl_str_mv Asención Diez, Matías Damián
Ebrecht, Ana Cristina
Martínez, Lucila Inés
Aleanzi, Mabel Cristina
Guerrero, Sergio Adrian
Ballicora, Miguel A.
Iglesias, Alberto Alvaro
author Asención Diez, Matías Damián
author_facet Asención Diez, Matías Damián
Ebrecht, Ana Cristina
Martínez, Lucila Inés
Aleanzi, Mabel Cristina
Guerrero, Sergio Adrian
Ballicora, Miguel A.
Iglesias, Alberto Alvaro
author_role author
author2 Ebrecht, Ana Cristina
Martínez, Lucila Inés
Aleanzi, Mabel Cristina
Guerrero, Sergio Adrian
Ballicora, Miguel A.
Iglesias, Alberto Alvaro
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv protein engineering
allosteric regulation
pyrophosphorylases evolution
UDP-glucose
ADP-glucose
topic protein engineering
allosteric regulation
pyrophosphorylases evolution
UDP-glucose
ADP-glucose
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans . In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.
Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Fil: Ballicora, Miguel A.. University Of Chicago; Estados Unidos de América;
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
description In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans . In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.
publishDate 2013
dc.date.none.fl_str_mv 2013-05
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/11336/1621
Asención Diez, Matías Damián; Ebrecht, Ana Cristina; Martínez, Lucila Inés; Aleanzi, Mabel Cristina; Guerrero, Sergio Adrian; et al.; A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators; Molecular Diversity Preservation International; International Journal Of Molecular Sciences; 14; 5; 5-2013; 9703-9721
1422-0067
url http://hdl.handle.net/11336/1621
identifier_str_mv Asención Diez, Matías Damián; Ebrecht, Ana Cristina; Martínez, Lucila Inés; Aleanzi, Mabel Cristina; Guerrero, Sergio Adrian; et al.; A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators; Molecular Diversity Preservation International; International Journal Of Molecular Sciences; 14; 5; 5-2013; 9703-9721
1422-0067
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.3390/ijms14059703
info:eu-repo/semantics/altIdentifier/url/http://www.mdpi.com/1422-0067/14/5/9703
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Molecular Diversity Preservation International
publisher.none.fl_str_mv Molecular Diversity Preservation International
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 13.22299

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