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dc.contributor.author
Alghamdi, Mashael A.  
dc.contributor.author
Hussien, Rania A.  
dc.contributor.author
Zheng, Yuanzhang  
dc.contributor.author
Patel, Hiral P.  
dc.contributor.author
Asención Diez, Matías Damián  
dc.contributor.author
Iglesias, Alberto Alvaro  
dc.contributor.author
Liu, Dali  
dc.contributor.author
Ballicora, Miguel A.  
dc.date.available
2023-09-27T17:03:22Z  
dc.date.issued
2022-07  
dc.identifier.citation
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  
dc.identifier.issn
0961-8368  
dc.identifier.uri
http://hdl.handle.net/11336/213298  
dc.description.abstract
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.  
dc.format
application/pdf  
dc.language.iso
eng  
dc.publisher
John Wiley & Sons  
dc.rights
info:eu-repo/semantics/openAccess  
dc.rights.uri
https://creativecommons.org/licenses/by/2.5/ar/  
dc.subject
ADP-GLUCOSE PYROPHOSPHORYLASE  
dc.subject
ALLOSTERIC REGULATION  
dc.subject
ENZYME ENGINEERING  
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GLYCOGEN BIOSYNTHESIS  
dc.subject.classification
Bioquímica y Biología Molecular  
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Ciencias Biológicas  
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CIENCIAS NATURALES Y EXACTAS  
dc.title
Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase  
dc.type
info:eu-repo/semantics/article  
dc.type
info:ar-repo/semantics/artículo  
dc.type
info:eu-repo/semantics/publishedVersion  
dc.date.updated
2023-07-07T20:49:27Z  
dc.journal.volume
31  
dc.journal.number
7  
dc.journal.pagination
1-13  
dc.journal.pais
Estados Unidos  
dc.journal.ciudad
Nueva York  
dc.description.fil
Fil: Alghamdi, Mashael A.. Al-imam Muhammad Ibn Saud Islamic University; Arabia Saudita. Loyola University Of Chicago; Estados Unidos  
dc.description.fil
Fil: Hussien, Rania A.. Loyola University Of Chicago; Estados Unidos. Al Baha University; Arabia Saudita  
dc.description.fil
Fil: Zheng, Yuanzhang. Loyola University Of Chicago; Estados Unidos  
dc.description.fil
Fil: Patel, Hiral P.. Loyola University Of Chicago; Estados Unidos  
dc.description.fil
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  
dc.description.fil
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  
dc.description.fil
Fil: Liu, Dali. Loyola University Of Chicago; Estados Unidos  
dc.description.fil
Fil: Ballicora, Miguel A.. Loyola University Of Chicago; Estados Unidos  
dc.journal.title
Protein Science  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/https://onlinelibrary.wiley.com/doi/10.1002/pro.4376  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.1002/pro.4376