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Exploring the active site of plant glutaredoxin by site-directed mutagenesis
Nicolas Rouhier1, Eric Gelhaye, Jean-Pierre Jacquot
1Unité Mixte de Recherches INRA, Université Henri-Poincaré, UMR IaM 1136, Faculté des Sciences, P.O. Box 239, 54506 Vandoeuve Cedex, France.
Abstract:
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.