Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.
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.
Couturier
Jeremy
J
Unité Mixte de Recherches 1136 UHP-INRA Interaction Arbres-Microorganismes, IFR 110 GEEF, Nancy Université, Faculté des Sciences, 54506 Vandoeuvre Cedex, France.
Koh
Cha San
CS
Zaffagnini
Mirko
M
Winger
Alison M
AM
Gualberto
Jose Manuel
JM
Corbier
Catherine
C
Decottignies
Paulette
P
Jacquot
Jean-Pierre
JP
Lemaire
Stéphane D
SD
Didierjean
Claude
C
Rouhier
Nicolas
N
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
D054477:Glutaredoxins; D010455:Peptides; D013438:Sulfhydryl Compounds; D005978:Glutathione