Characterization of sorbitol-6-P dehydrogenase from leaves

FIGUEROA CM; IGLESIAS AA

Mar del Plata

Congreso; XLIII Reunión Anual de la SAIB; 2007

SAIB

Sorbitol is the major photosynthetic end-product in many crop trees, including apple and peach. Despite this relevance, relatively few studies have been performed on the kinetic, regulatory and structural characterization of the enzymes involved in sorbitol synthesis in plants. Recently, we developed the recombinant expression of sorbitol-6P dehydrogenase (S6PDHase, EC 1.1.1.200) from apple and peach leaves. In order to improve the level of expression and purification, we placed a His-tag in the protein N-term instead of the C-term previously utilized. The specific activity of the N-term tagged enzyme is near 50-fold higher compared with the C-term tagged one. This result is in good agreement with a homology model based in the crystal structure of xylose reductase from Candida tenuis, a member of the group 2 of the aldo-keto reductase superfamily. According to the model, the C-term appears to be involved in the interaction between both subunits in the dimer. Oxidation assays showed complete inactivation of S6PDHase after incubation with 2 mM diamide. The oxidation is prevented by NADPH, but glucose-6P showed no effect. Results suggest the relevance of cysteine residues in catalysis and the interaction of the enzyme with substrates. Also, they establish models to understand structure-function relationships in a key enzyme for carbon partitioning in plants accumulating sugar-alcohols.