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Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.

Identifieur interne : 003474 ( Main/Exploration ); précédent : 003473; suivant : 003475

Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.

Auteurs : Jeremy Couturier [France] ; Cha San Koh ; Mirko Zaffagnini ; Alison M. Winger ; Jose Manuel Gualberto ; Catherine Corbier ; Paulette Decottignies ; Jean-Pierre Jacquot ; Stéphane D. Lemaire ; Claude Didierjean ; Nicolas Rouhier

Source :

RBID : pubmed:19158074

Descripteurs français

English descriptors

Abstract

Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol (28)WCSYS(32) active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys(87)) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys(87) exhibits a certain reactivity, since its pK(a) is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys(29)) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation.

DOI: 10.1074/jbc.M807998200
PubMed: 19158074
PubMed Central: PMC2666582


Affiliations:


Links toward previous steps (curation, corpus...)


Le document en format XML

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<div type="abstract" xml:lang="en">Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol (28)WCSYS(32) active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys(87)) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys(87) exhibits a certain reactivity, since its pK(a) is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys(29)) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation.</div>
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