Exploring the active site of plant glutaredoxin by site-directed mutagenesis.
Six mutants (Y26A, C27S, Y29F, Y29P, C30S and Y26W/Y29P) have been engineered in order to explore the active site of poplar glutaredoxin (Grx) (Y26CPYC30). The cysteinic mutants indicate that Cys 27 is the primary nucleophile. Phe is a good substitute for Tyr 29, but the Y29P mutant was inactive. The Y26A mutation caused a moderate loss of activity. The YCPPC and WCPPC mutations did not improve the reactivity of Grx with the chloroplastic NADP-malate dehydrogenase, a well known target of thioredoxins (Trxs). The results are discussed in relation with the known biochemical properties of Grx and Trx.
Rouhier
Nicolas
N
Unité Mixte de Recherches INRA, Université Henri-Poincaré, UMR IaM 1136, Faculté des Sciences, P.O. Box 239, 54506 Vandoeuve Cedex, France.
Gelhaye
Eric
E
Jacquot
Jean-Pierre
JP
D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003683:Dehydroascorbic Acid / Q000378:metabolism / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D000431:Ethanol / Q000031:analogs & derivatives* / Q000378:metabolism; D054477:Glutaredoxins; D019684:Magnoliopsida / Q000235:genetics / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014197:Trees / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D024322:Amino Acids, Aromatic; D004220:Disulfides; D054477:Glutaredoxins; D010940:Plant Proteins; D011506:Proteins; D000431:Ethanol; D014443:Tyrosine; C031319:2-hydroxyethyl disulfide; D009249:NADP; D010088:Oxidoreductases; D008291:Malate Dehydrogenase; D003683:Dehydroascorbic Acid