Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae

Detalhes bibliográficos
Autor(a) principal: Discola, Karen Fulan
Data de Publicação: 2009
Outros Autores: de Oliveira, Marcos Antonio [UNESP], Rosa Cussiol, José Renato, Monteiro, Gisele, Bárcena, José Antonio, Porras, Pablo, Padilla, C. Alicia, Guimarães, Beatriz Gomes, Netto, Luis Eduardo Soares
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://dx.doi.org/10.1016/j.jmb.2008.10.055
http://hdl.handle.net/11449/225384
Resumo: Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower KM for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pKa of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pKa values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 Å, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity. © 2008 Elsevier Ltd. All rights reserved.
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spelling Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiaedisulfideglutaredoxinglutathioneSaccharomyces cerevisiaeX-ray structureGlutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower KM for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pKa of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pKa values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 Å, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity. © 2008 Elsevier Ltd. All rights reserved.Departamento de Bioquímica Instituto de Biologia Universidade Estadual de Campinas, 13083-970 CampinasDepartamento de Genética e Biologia Evolutiva Instituto de Biociências Universidade de São Paulo, 05508-900 São PauloDepartamento de Biologia Universidade Estadual Paulista, 11330-900 São VicenteDepartamento de Bioquímica y Biología Molecular Universidad de Córdoba, 14071 CórdobaLaboratório Nacional de Luz Síncrotron, CampinasDepartamento de Biologia Universidade Estadual Paulista, 11330-900 São VicenteUniversidade Estadual de Campinas (UNICAMP)Universidade de São Paulo (USP)Universidade Estadual Paulista (UNESP)Universidad de CórdobaLaboratório Nacional de Luz SíncrotronDiscola, Karen Fulande Oliveira, Marcos Antonio [UNESP]Rosa Cussiol, José RenatoMonteiro, GiseleBárcena, José AntonioPorras, PabloPadilla, C. AliciaGuimarães, Beatriz GomesNetto, Luis Eduardo Soares2022-04-28T20:48:13Z2022-04-28T20:48:13Z2009-01-23info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article889-901http://dx.doi.org/10.1016/j.jmb.2008.10.055Journal of Molecular Biology, v. 385, n. 3, p. 889-901, 2009.0022-2836http://hdl.handle.net/11449/22538410.1016/j.jmb.2008.10.0552-s2.0-58149123295Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengJournal of Molecular Biologyinfo:eu-repo/semantics/openAccess2022-04-28T20:48:13Zoai:repositorio.unesp.br:11449/225384Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462022-04-28T20:48:13Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
title Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
spellingShingle Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
Discola, Karen Fulan
disulfide
glutaredoxin
glutathione
Saccharomyces cerevisiae
X-ray structure
title_short Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
title_full Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
title_fullStr Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
title_full_unstemmed Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
title_sort Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
author Discola, Karen Fulan
author_facet Discola, Karen Fulan
de Oliveira, Marcos Antonio [UNESP]
Rosa Cussiol, José Renato
Monteiro, Gisele
Bárcena, José Antonio
Porras, Pablo
Padilla, C. Alicia
Guimarães, Beatriz Gomes
Netto, Luis Eduardo Soares
author_role author
author2 de Oliveira, Marcos Antonio [UNESP]
Rosa Cussiol, José Renato
Monteiro, Gisele
Bárcena, José Antonio
Porras, Pablo
Padilla, C. Alicia
Guimarães, Beatriz Gomes
Netto, Luis Eduardo Soares
author2_role author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universidade Estadual de Campinas (UNICAMP)
Universidade de São Paulo (USP)
Universidade Estadual Paulista (UNESP)
Universidad de Córdoba
Laboratório Nacional de Luz Síncrotron
dc.contributor.author.fl_str_mv Discola, Karen Fulan
de Oliveira, Marcos Antonio [UNESP]
Rosa Cussiol, José Renato
Monteiro, Gisele
Bárcena, José Antonio
Porras, Pablo
Padilla, C. Alicia
Guimarães, Beatriz Gomes
Netto, Luis Eduardo Soares
dc.subject.por.fl_str_mv disulfide
glutaredoxin
glutathione
Saccharomyces cerevisiae
X-ray structure
topic disulfide
glutaredoxin
glutathione
Saccharomyces cerevisiae
X-ray structure
description Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower KM for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pKa of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pKa values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 Å, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity. © 2008 Elsevier Ltd. All rights reserved.
publishDate 2009
dc.date.none.fl_str_mv 2009-01-23
2022-04-28T20:48:13Z
2022-04-28T20:48:13Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.1016/j.jmb.2008.10.055
Journal of Molecular Biology, v. 385, n. 3, p. 889-901, 2009.
0022-2836
http://hdl.handle.net/11449/225384
10.1016/j.jmb.2008.10.055
2-s2.0-58149123295
url http://dx.doi.org/10.1016/j.jmb.2008.10.055
http://hdl.handle.net/11449/225384
identifier_str_mv Journal of Molecular Biology, v. 385, n. 3, p. 889-901, 2009.
0022-2836
10.1016/j.jmb.2008.10.055
2-s2.0-58149123295
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Journal of Molecular Biology
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 889-901
dc.source.none.fl_str_mv Scopus
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
collection Repositório Institucional da UNESP
repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
repository.mail.fl_str_mv
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