title: Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.
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.
authors.affiliation: Unité Mixte de Recherches 1136 UHP-INRA Interaction Arbres-Microorganismes, IFR 110 GEEF, Nancy Université, Faculté des Sciences, 54506 Vandoeuvre Cedex, France.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Couturier
authors.forename: Cha San
authors.initials: CS
authors.lastname: Koh
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.forename: Alison M
authors.initials: AM
authors.lastname: Winger
authors.forename: Jose Manuel
authors.initials: JM
authors.lastname: Gualberto
authors.forename: Catherine
authors.initials: C
authors.lastname: Corbier
authors.forename: Paulette
authors.initials: P
authors.lastname: Decottignies
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Jacquot
authors.forename: Stéphane D
authors.initials: SD
authors.lastname: Lemaire
authors.forename: Claude
authors.initials: C
authors.lastname: Didierjean
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D054477:Glutaredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D032107:Populus / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D017186:Titrimetry
chemical_list: D054477:Glutaredoxins; D010455:Peptides; D013438:Sulfhydryl Compounds; D005978:Glutathione