Posttranslational modification of UDP-glucose dehydrogenase

The products from photosynthesis can be used for energy metabolism, for growth and for the formation of storage compounds in plants. The partitioning of carbohydrates for the synthesis of cell wall polymers, required for growth, will be analysed in this proposal. The enzyme UDP-glucose dehydrogenase is in the centre, because its product contributes to about half of the cell wall biomass from leaves and to about one third of the wood biomass. The enzyme is posttranslationally modified as we could show by mass spectroscopy. The relevance of this modification for enzyme activity and regulation of the carbohydrate partitioning will be analysed in detail in this project. The major competitor enzyme is sucrose-phosphate-synthase, which is regulated in its activity by protein phosphorylation. Therefore we expect a regulatory function of the posttranslational modification of UDP-glucose dehydrogenase. The function of the protein modification for carbohydrate partitioning into cell walls can be analysed with our toolbox (monoclonal antibodies; mutants and transgenic lines; biochemical analytics). The results are important for the breeding of energy crops, which predominantly use plant cell walls for energy conversion.

Biopolymers from plant cell walls are mainly synthesized from activated sugars (UDP- sugars). The enzyme UDP-glucose dehydrogenase (UGD) is responsible for more than half of the cell wall biomass in plant leaves. In plants, UGD has an additional peptide loop at the protein surface which is absent in bacterial or human sequences. We showed that a serine residue within this surface exposed loop can be phosphorylated. We identified MAP-kinase 3 as the responsible kinase for phosphorylation. Recombinant UGD protein can be quantitatively phosphorylated by activated MAP-kinase 3. The enzyme UGD uses UDP- glucose as substrate and is therefore a competitor for sucrose formation in plants. The competing enzyme sucrose-phosphate synthase is inactivated by phosphorylation which inspired us to suggest a similar regulation of UGD. Purified phosphorylated UGD however shows no reduction in enzyme activity compared to the non-phosphorylated control. The enzyme UGD is located in the cytoplasm but the product of the enzymatic reaction is needed in the Golgi apparatous. Using fusions with fluorescent proteins we analyzed the intracellular localization. The assumption that phosphorylation of UGD causes a relocalization of the enzyme to the sink Golgi-apparatous could not be confirmed.