Koh CS, Navrot N, Didierjean C, Rouhier N, Hirasawa M, Knaff DB, Wingsle G, Samian R, Jacquot J-P, Corbier C, Gelhaye E
An atypical catalytic mechanism involving three cysteines of thioredoxin
Journal of Biological Chemistry: 2008 283:23062-23072

Unlike other thioredoxins h characterized so far, a poplar thioredoxinof the h type, PtTrxh4, is reduced by glutathione and glutaredoxin(Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 containsthree cysteines: one localized in an N-terminal extension (Cys4)and two (Cys58 and Cys61) in the classical thioredoxin activesite (57WCGPC61). The property of a mutant in which Cys58 wasreplaced by serine demonstrates that it is responsible for theinitial nucleophilic attack during the catalytic cycle. Theobservation that the C4S mutant is inactive in the presenceof Grx but fully active when dithiothreitol is used as a reductantindicates that Cys4 is required for the regeneration of PtTrxh4by Grx. Biochemical and x-ray crystallographic studies indicatethat two intramolecular disulfide bonds involving Cys58 canbe formed, linking it to either Cys61 or Cys4. We propose thusa four-step disulfide cascade mechanism involving the transientglutathionylation of Cys4 to convert this atypical thioredoxinh back to its active reduced form.

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