Mannitol Synthesis in Higher Plants Evidence for the Role and Characterization of a NADPH-Dependent Mannose 6-Phosphate Reductase

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase 14 C-radiolabeling studies with the mannitol-synthesizing species, celery ( Apium graveolens L.) and privet ( Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K m (M6P) was 15.8 millimolar, but the apparent K m (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg 2+ , Ca 2+ , Zn 2+ , and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme9s activity. In vivo radiolabeling results and the enzyme9s kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.