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.
Koh
Cha San
CS
Equipe Biocristallographie, UMR 7036 CNRS-Université Henri Poincaré, URAFPA, Equipe PB2P, Faculté des Sciences et Techniques, Nancy Université, BP 239, 54506 Vandoeuvre Cedex France.
Navrot
Nicolas
N
Didierjean
Claude
C
Rouhier
Nicolas
N
Hirasawa
Masakazu
M
Knaff
David B
DB
Wingsle
Gunnar
G
Samian
Razip
R
Jacquot
Jean-Pierre
JP
Corbier
Catherine
C
Gelhaye
Eric
E
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