Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
Autor(a) principal: | |
---|---|
Data de Publicação: | 2009 |
Outros Autores: | , , , , , , , |
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. |
id |
UNSP_0d77e1f7e6b1a963932a22e79cfb6dc4 |
---|---|
oai_identifier_str |
oai:repositorio.unesp.br:11449/225384 |
network_acronym_str |
UNSP |
network_name_str |
Repositório Institucional da UNESP |
repository_id_str |
2946 |
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 |
|
_version_ |
1799965748430372864 |