Phosphorylation of Thr257 inhibits aldose-6-phosphate reductase from peach leaves

ROJAS, BRUNO E.; FIGUEROA, CARLOS M.; HARTMAN, MATÍAS D.; IGLESIAS, ALBERTO A.; BALLICORA, MIGUEL A.

Buenos Aires

Congreso; Reunión conjunta de sociedades de biociencias; 2017

SAIB y otras

In addition to sucrose and starch, glucitol (a sugar alcohol)is a primary photosynthate in a considerable number of agronomicallyimportant plant species. Glucitol is produced in matureleaves from glucose 6-phosphate by the combined action of theNADPH-dependent aldose-6-phosphate reductase (Ald6PRase,EC 1.1.1.200) and a specific phosphatase. We recently found thatactivity of Ald6PRase from peach (Prunus persica) leaves (PpeAld-6PRase) is inhibited by hexose-phosphates, Pi and oxidants. In thiswork, we show that PpeAld6PRase from mature leaves is phosphorylatedin vivo. Treatment of the phosphorylated enzyme withalkaline phosphatase increased the activity 3-fold. RecombinantPpeAld6PRase was phosphorylated in vitro by a partially purified extractfrom peach leaves, but also by recombinant Mg2+- and Ca2+-dependentprotein kinases. Using in silico approaches we identifieda highly conserved Thr residue as a putative phosphorylation site.The phosphomimetic T257D mutant had negligible catalytic activityand was less phosphorylated than the WT. Because Thr257 is locatedin the predicted NADPH-binding loop and presence of NADPHprevents phosphorylation of the WT enzyme, we hypothesized thatphosphorylation of this residue prevents cofactor binding. Dye-bindingcoupled to temperature-dependent denaturing assays showedimpaired NADPH-binding to the T257D mutant. Our results stronglysuggest that phosphorylation regulates Ald6PRase activity and, as aconsequence, glucitol levels in peach leaves.