Project Details
Projekt
Structural and biochemical analyses of the disproportionating isozyme 2 (DPE2)
Subject Area
Structural Biology
Plant Physiology
Plant Physiology
Term
from 2013 to 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 239353016
Starch metabolism in plants is complex and combines several pathways for either storing or metabolizing sugars. Moreover, starch mobilization in the mesophyll cell has to integrate over various plastids, which differ in size as well as starch level, to export neutral sugars into a uniform reaction volume, i.e. the cytosol. The key player in this complex metabolic network is the disproportionating isozyme 2 (DPE2), which metabolises the disaccharide beta-maltose, the quantitatively dominant product of transitory starch degradation during the night. DPE2 transfers one glucosyl residue from beta-maltose to cytosolic heteroglycans and releases the other hexosyl moiety as a free glucose.The DPE2 monomer is a multi-modular protein with a molecular weight of approximately 100 kDa. In its functional state, however, DPE2 has a more complex quaternary structure. DPE2 from several higher plant species such as Arabidopsis thaliana, Solanum tuberosum and Zea mays as well as recombinant DPE2 occurs in a high-molecular weight state of approximately 1 MDa (state I). Additionally, recombinant DPE2 exists in an smaller oligomeric state having an apparent molecular weight of approximately 300 kDa (state II), which was also found in tissues from Arabidopsis. Moreover, DEP2 state I contains endogenous carbohydrates which might act as internal acceptors during the glucosyl transfer reaction. However, the precise reaction mechanism of this crucial step in starch degradation is poorly understood.Within this project, we aim to analyse the structural organization and the reaction mechanism of DPE2 combining biochemical, kinetic and molecular biological approaches with cryo-electron microscopy and protein X-ray crystallography. Firstly, we want to elucidate the composition of DPE2-complexes from different plants at different physiological states. Further, we want to characterize the physiological importance of different oligomeric DPE2 states and study the functional role of endogenous glycan components present in DPE2-complexes. Supplementary, we want to combine cryo-electron microscopy and X-ray diffraction techniques to analyze the structural organization of DPE2 complexes, which is essential to understand this central element of the plant sugar metabolism at a molecular level.
DFG Programme
Research Grants
