An atypical catalytic mechanism involving three cysteines of thioredoxin.
Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys(4)) and two (Cys(58) and Cys(61)) in the classical thioredoxin active site ((57)WCGPC(61)). The property of a mutant in which Cys(58) was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys(4) is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys(58) can be formed, linking it to either Cys(61) or Cys(4). We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys(4) to convert this atypical thioredoxin h back to its active reduced form.
Equipe Biocristallographie, UMR 7036 CNRS-Université Henri Poincaré, URAFPA, Equipe PB2P, Faculté des Sciences et Techniques, Nancy Université, BP 239, 54506 Vandoeuvre Cedex France.
Cha San
CS
Koh
Nicolas
N
Navrot
Claude
C
Didierjean
Nicolas
N
Rouhier
Masakazu
M
Hirasawa
David B
DB
Knaff
Gunnar
G
Wingsle
Razip
R
Samian
Jean-Pierre
JP
Jacquot
Catherine
C
Corbier
Eric
E
Gelhaye
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D054477:Glutaredoxins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism
D054477:Glutaredoxins; D010940:Plant Proteins; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine; D004229:Dithiothreitol