Structural and biochemical analyses of the disproportionating isozyme 2 (DPE2)

植物中的淀粉代谢很复杂，结合了多种储存或代谢糖的途径。此外，叶肉细胞中的淀粉动员必须在大小和淀粉水平不同的各种质体上整合，以将中性糖输出到均匀的反应体积，即细胞质中。在这个复杂的代谢网络中，关键的参与者是分解同工酶2（DPE 2），它代谢二糖β-麦芽糖，这是夜间短暂淀粉降解的定量主导产物。DPE 2将一个葡糖基残基从β-麦芽糖转移到胞质杂聚糖，并释放另一个己糖基部分作为游离葡萄糖APE 2单体是分子量约为IOOkDa的多模块蛋白质。然而，在其功能状态下，DPE 2具有更复杂的四级结构。来自几种高等植物物种如拟南芥、马铃薯和玉米的DPE 2以及重组DPE 2以约1 MDa的高分子量状态（状态I）存在。此外，重组DPE 2以较小的寡聚体状态存在，具有约300 kDa的表观分子量（状态II），这也在拟南芥的组织中发现。此外，DEP 2状态I含有内源性碳水化合物，其可能在葡萄糖基转移反应期间充当内部受体。然而，在淀粉降解的这一关键步骤的精确反应机理知之甚少。在这个项目中，我们的目标是分析结构组织和DPE 2的反应机理结合生化，动力学和分子生物学方法与冷冻电子显微镜和蛋白质X射线晶体学。首先，我们要阐明的DPE 2-复合物的组成，从不同的植物在不同的生理状态。此外，我们希望表征不同寡聚体DPE 2状态的生理重要性，并研究DPE 2复合物中存在的内源性聚糖组分的功能作用。作为补充，我们希望结合联合收割机和X射线衍射技术来分析DPE 2复合物的结构组织，这对于在分子水平上理解植物糖代谢的核心元素是必不可少的。

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.