Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase

Autores
Alghamdi, Mashael A.; Hussien, Rania A.; Zheng, Yuanzhang; Patel, Hiral P.; Asención Diez, Matías Damián; Iglesias, Alberto Alvaro; Liu, Dali; Ballicora, Miguel A.
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The allosteric regulation of ADP–glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.
Fil: Alghamdi, Mashael A.. Al-imam Muhammad Ibn Saud Islamic University; Arabia Saudita. Loyola University Of Chicago; Estados Unidos
Fil: Hussien, Rania A.. Loyola University Of Chicago; Estados Unidos. Al Baha University; Arabia Saudita
Fil: Zheng, Yuanzhang. Loyola University Of Chicago; Estados Unidos
Fil: Patel, Hiral P.. Loyola University Of Chicago; Estados Unidos
Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Loyola University Of Chicago; Estados Unidos
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Liu, Dali. Loyola University Of Chicago; Estados Unidos
Fil: Ballicora, Miguel A.. Loyola University Of Chicago; Estados Unidos
Materia
ADP-GLUCOSE PYROPHOSPHORYLASE
ALLOSTERIC REGULATION
ENZYME ENGINEERING
GLYCOGEN BIOSYNTHESIS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/213298
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/213298
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylaseAlghamdi, Mashael A.Hussien, Rania A.Zheng, YuanzhangPatel, Hiral P.Asención Diez, Matías DamiánIglesias, Alberto AlvaroLiu, DaliBallicora, Miguel A.ADP-GLUCOSE PYROPHOSPHORYLASEALLOSTERIC REGULATIONENZYME ENGINEERINGGLYCOGEN BIOSYNTHESIShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The allosteric regulation of ADP–glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.Fil: Alghamdi, Mashael A.. Al-imam Muhammad Ibn Saud Islamic University; Arabia Saudita. Loyola University Of Chicago; Estados UnidosFil: Hussien, Rania A.. Loyola University Of Chicago; Estados Unidos. Al Baha University; Arabia SauditaFil: Zheng, Yuanzhang. Loyola University Of Chicago; Estados UnidosFil: Patel, Hiral P.. Loyola University Of Chicago; Estados UnidosFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Loyola University Of Chicago; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Liu, Dali. Loyola University Of Chicago; Estados UnidosFil: Ballicora, Miguel A.. Loyola University Of Chicago; Estados UnidosJohn Wiley & Sons2022-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/213298Alghamdi, Mashael A.; Hussien, Rania A.; Zheng, Yuanzhang; Patel, Hiral P.; Asención Diez, Matías Damián; et al.; Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase; John Wiley & Sons; Protein Science; 31; 7; 7-2022; 1-130961-8368CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://onlinelibrary.wiley.com/doi/10.1002/pro.4376info:eu-repo/semantics/altIdentifier/doi/10.1002/pro.4376info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:45:44Zoai:ri.conicet.gov.ar:11336/213298instacron: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:34982025-09-29 10:45:44.288CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
title Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
spellingShingle Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
Alghamdi, Mashael A.
ADP-GLUCOSE PYROPHOSPHORYLASE
ALLOSTERIC REGULATION
ENZYME ENGINEERING
GLYCOGEN BIOSYNTHESIS
title_short Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
title_full Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
title_fullStr Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
title_full_unstemmed Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
title_sort Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase
dc.creator.none.fl_str_mv Alghamdi, Mashael A.
Hussien, Rania A.
Zheng, Yuanzhang
Patel, Hiral P.
Asención Diez, Matías Damián
Iglesias, Alberto Alvaro
Liu, Dali
Ballicora, Miguel A.
author Alghamdi, Mashael A.
author_facet Alghamdi, Mashael A.
Hussien, Rania A.
Zheng, Yuanzhang
Patel, Hiral P.
Asención Diez, Matías Damián
Iglesias, Alberto Alvaro
Liu, Dali
Ballicora, Miguel A.
author_role author
author2 Hussien, Rania A.
Zheng, Yuanzhang
Patel, Hiral P.
Asención Diez, Matías Damián
Iglesias, Alberto Alvaro
Liu, Dali
Ballicora, Miguel A.
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv ADP-GLUCOSE PYROPHOSPHORYLASE
ALLOSTERIC REGULATION
ENZYME ENGINEERING
GLYCOGEN BIOSYNTHESIS
topic ADP-GLUCOSE PYROPHOSPHORYLASE
ALLOSTERIC REGULATION
ENZYME ENGINEERING
GLYCOGEN BIOSYNTHESIS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The allosteric regulation of ADP–glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.
Fil: Alghamdi, Mashael A.. Al-imam Muhammad Ibn Saud Islamic University; Arabia Saudita. Loyola University Of Chicago; Estados Unidos
Fil: Hussien, Rania A.. Loyola University Of Chicago; Estados Unidos. Al Baha University; Arabia Saudita
Fil: Zheng, Yuanzhang. Loyola University Of Chicago; Estados Unidos
Fil: Patel, Hiral P.. Loyola University Of Chicago; Estados Unidos
Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Loyola University Of Chicago; Estados Unidos
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Liu, Dali. Loyola University Of Chicago; Estados Unidos
Fil: Ballicora, Miguel A.. Loyola University Of Chicago; Estados Unidos
description The allosteric regulation of ADP–glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.
publishDate 2022
dc.date.none.fl_str_mv 2022-07
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/11336/213298
Alghamdi, Mashael A.; Hussien, Rania A.; Zheng, Yuanzhang; Patel, Hiral P.; Asención Diez, Matías Damián; et al.; Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase; John Wiley & Sons; Protein Science; 31; 7; 7-2022; 1-13
0961-8368
CONICET Digital
CONICET
url http://hdl.handle.net/11336/213298
identifier_str_mv Alghamdi, Mashael A.; Hussien, Rania A.; Zheng, Yuanzhang; Patel, Hiral P.; Asención Diez, Matías Damián; et al.; Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase; John Wiley & Sons; Protein Science; 31; 7; 7-2022; 1-13
0961-8368
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://onlinelibrary.wiley.com/doi/10.1002/pro.4376
info:eu-repo/semantics/altIdentifier/doi/10.1002/pro.4376
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv John Wiley & Sons
publisher.none.fl_str_mv John Wiley & Sons
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 13.070432

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