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

歧化同工酶 2 (DPE2) 的结构和生化分析

基本信息

批准号：
239353016
负责人：
Dr. Thorsten Mielke
金额：
--
依托单位：
Max-Planck-Institut für molekulare Genetik (MPIMG)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2016-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/239353016?language=en
关键词：
Structural biochemical analyses disproportionating isozyme

项目摘要

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.

植物中的淀粉代谢是复杂的，并结合了几条储存或代谢糖的途径。此外，叶肉细胞中的淀粉动员必须整合到不同大小和不同淀粉水平的各种质体上，以将中性糖输出到一个统一的反应体积，即细胞质。在这个复杂的代谢网络中，关键的参与者是不成比例同工酶2(DPE2)，它代谢二糖β-麦芽糖，这是夜间短暂淀粉降解的数量优势产物。DPE2将一个葡萄糖残基从β-麦芽糖转移到胞质杂多糖中，并释放另一个己糖部分作为游离葡萄糖。DPE2单体是一种多模块蛋白质，分子量约为100 kDa。然而，在其功能状态下，DPE2具有更复杂的四元结构。来自拟南芥、茄子和玉米等高等植物的DPE2以及重组的DPE2以约1丙二醛的高分子量状态存在(状态I)。此外，重组DPE2以较小的寡聚状态存在，其表观分子量约为300 kDa(状态II)，这也在拟南芥组织中发现。此外，DEP2状态I含有内源性碳水化合物，这些碳水化合物可能在葡萄糖转移反应中作为内部受体。然而，对淀粉降解这一关键步骤的确切反应机理还不甚了解。在这个项目中，我们旨在结合生化、动力学和分子生物学方法，结合冷冻电子显微镜和蛋白质X-射线结晶学分析DPE2的结构组成和反应机理。首先，我们想要阐明不同植物在不同生理状态下的DPE2-复合体的组成。此外，我们想要表征不同寡聚体DPE2状态的生理重要性，并研究存在于DPE2-复合体中的内源性多糖成分的功能作用。作为补充，我们希望结合低温电子显微镜和X射线衍射技术来分析DPE2复合体的结构组织，这对于在分子水平上理解这一植物糖代谢的中心元件是必不可少的。