title: Glutaredoxin-dependent peroxiredoxin from poplar: protein-protein interaction and catalytic mechanism.
abstract: Recently, a poplar phloem peroxiredoxin (Prx) was found to accept both glutaredoxin (Grx) and thioredoxin (Trx) as proton donors. To investigate the catalytic mechanism of the Grx-dependent reduction of hydroperoxides catalyzed by Prx, a series of cysteinic mutants was constructed. Mutation of the most N-terminal conserved cysteine of Prx (Cys-51) demonstrates that it is the catalytic one. The second cysteine (Cys-76) is not essential for peroxiredoxin activity because the C76A mutant retained approximately 25% of the wild type Prx activity. Only one cysteine of the Grx active site (Cys-27) is essential for peroxiredoxin catalysis, indicating that Grx can act in this reaction either via a dithiol or a monothiol pathway. The creation of covalent heterodimers between Prx and Grx mutants confirms that Prx Cys-51 and Grx Cys-27 are the two residues involved in the catalytic mechanism. The integration of a third cysteine in position 152 of the Prx, making it similar in sequence to the Trx-dependent human Prx V, resulted in a protein that had no detectable activity with Grx but kept activity with Trx. Based on these experimental results, a catalytic mechanism is proposed to explain the Grx- and Trx-dependent activities of poplar Prx.
authors.affiliation: Unité mixte de recherche IaM INRA-UHP Nancy I, Biochimie et Biologie Moléculaire Végétales, Université Henri Poincaré, 54506 Vandoeuvre Cedex, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
authors.forename: Eric
authors.initials: E
authors.lastname: Gelhaye
authors.forename: Jean Pierre
authors.initials: JP
authors.lastname: Jacquot
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; C516005:GLRX protein, human; D054477:Glutaredoxins; D011506:Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D010544:Peroxidases; D054464:Peroxiredoxins; D003545:Cysteine; D010100:Oxygen