Mechanism for the regulation of wheat non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase.

GUERRERO, SERGIO ADRIÁN; PIATTONI VANESA,; IGLESIAS ALBERTO,; BUSTOS DIEGO,

Rosario

Congreso; 42th Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2006

SAIB

Mechanisms for the regulation of wheat non-phosphorylating glyceraldehyde-3-phospate dehydrogenase Claudia V Piattoni, Diego M. Bustos*, Sergio A Guerrero, Alberto A Iglesias Lab. Enzimología Molecular y Lab. Bioquímica Microbiana, Fac. Bioq. y Cs. Biol., UNl, Santa Fe.*IIB-INTECH, Chascomús. E-mail: piattoni@fbcb.unl.edu.ar Non-phosphorylating, NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.1.9; NP-GaPDHase) is a cytosolic enzyme found photosynthetic eukaryotes. The enzyme is involved in a shuttle system exporting NADPH from chloroplasts to cytosol. In heterotrophic plant cells its function is less clear and it could couple NADPH production to glycolysis. In wheat endosperm (but not in leaves) NP-GaPDHase is post-translationally modified by phosphorylation, after which it interacts with 14‑3-3 proteins. We heterologously expressed the gene encoding a NP-GaPDHase from wheat (NCBI; NºAcc:AF521191) and a NP-GaPDHaseS404A mutant form, and we purified both enzymes to a high degree. NP-GaPDHase was phosphorylated by wheat endosperm extracts under reaction conditions specific for SNF1-related protein kinases, but not for another kinases (SOS2, GSK-3, and MAPK). The mutant S404A enzyme exhibited higher specific activity and was recalcitrant to be phosphorylated under the different conditions. These results reinforce the structural model predicting Ser404 as a regulatory phosphorylation site in the enzyme. NP-GaPDHase was also inactivated by the thiol oxidative chemicals DTNB and Diamide. This inactivation was reverted by DTT and reduced thioredoxin, suggesting that NP-GaPDHase could be regulated by redox systems operating in the cytoplasm of plant cells. (Granted by: UNL, CAI+D 2006, ANPCyT, IM40 2000, PICT´03 01-14733, PAV´03 137.)