Cloning, expression and characterization of sorbitol-6-P dehydrogenase from apple and peach leaves

FIGUEROA CM; IGLESIAS AA

Rosario

Congreso; XLII Reunión anual de la SAIB; 2006

SAIB

In addition to sucrose and starch, sorbitol is a major photosynthetic end-product in many economically important fruit-bearing tree species, including apple and peach. Despite this relevance, relatively few studies have been performed on the regulation of sorbitol synthesis. Recently, two enzymes from mature apple leaves were purified and characterized, demonstrating that sorbitol formation is catalysed by the sequential action of sorbitol-6-P dehydrogenase (S6PDHase; Glc6P + NADPH ↔ Sor6P + NADP+), and sorbitol-6-P phosphatase (Sor6P + H2O → sorbitol + Pi). The sugar-alcohol is translocated from mature leaves to growing tissues, such as fruits and young leaves, where it may serve as osmoprotectant, as it occurs with mannitol in celery. In our aim to elucidate the structure to function relationships of S6PDHase, we have cloned the genes encoding for this enzyme from apple and peach leaves. The recombinant proteins were expressed in Escherichia coli cells, purified and characterized. The results obtained are in good agreement with the previously reported data. The recombinant enzymes were specific to reversibly reduce Glc6P utilizing NADPH. Also, a 3D model of S6PDHase was built with the SWISS-MODEL server, using the human aldose reductase as template. To the best of our knowledge, this is the first time that S6PDHase is successfully expressed in a prokaryotic system.