Detailed analyses of starch granule surface structure alterations

淀粉颗粒表面结构变化的详细分析

• 批准号: 417600839
• 负责人: Professor Dr. Joerg Fettke
• 金额: --
• 依托单位: Institut für Biochemie und Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/417600839?language=en
• 关键词: Detailed; analyses; starch; granule; surface

Starch degradation is controlled by a phosphorylation/dephosphorylation cycle in which first the surface of the starch granules is phosphosphorylated by α-glucan, water dikinase (GWD) and phosphoglucan water dikinase (PWD). This decreases the surface crystallinity and transforms the highly ordered glucan chains of amylopectin into a solubilized state. These solubilized glucan chains provide suitable substrates for hydrolases such as β-amylases and isoamylases, with the orchestrated action of the phosphatases SEX4 and LSF2 being obligatory. To date, little is known about the regulation of these processes at the starch granule surface. At the same time, it also remains unclear in detail how the surface structures/properties influence the enzymes involved in starch metabolism and, vice versa, how these surface properties/morphologies of the granules are altered in detail by the action of specific proteins/enzymes. For the recently identified protein ESV1, which is essential for the rate of starch degradation during the dark phase, it was shown in in vitro studies that it alters dikinase-mediated phosphate incorporation. Similarly, this effect occurred with the homolog LESV. It was found that these effects were not caused by protein-protein interactions, but rather by the binding of ESV1 and LESV at specific structures at the starch granule surface. It was shown that the two proteins differ in their glucan binding specificity, with ESV1 preferentially binding to amylopectin and LESV to amylose. An influence of ESV1 and LESV on other enzymes involved in starch metabolism such as BAM, ISA, SEX4, PHS1, and SS1 was also confirmed. However, the exact molecular background is unclear. In this proposal, this mechanism will be analysed in detail using different experimental approaches and considering the following main objectives. (1) Identifying the glucan structures bound by proteins ESV1 and LESV and which protein region is responsible for the observed effects on enzymes involved in starch metabolism. (2) The analysis of other enzymes/proteins involved in starch metabolism that are affected by ESV1 and LESV. (3) Investigating the phosphorylation pattern of GWD and PWD from different species and the influence of ESV1 and LESV on this pattern. (4) The analysis of the surface structural characteristics of starch granules in different mutants during the diurnal cycle and aging process, and how these structural properties determine granule characteristics as size, size distribution and morphology.

淀粉降解受磷酸化/去磷酸化循环控制，其中淀粉颗粒的表面首先被α-葡聚糖、水双激酶（GWD）和磷酸葡聚糖水双激酶（PWD）磷酸化。这降低了表面结晶度，并将支链淀粉的高度有序的葡聚糖链转化为溶解状态。这些溶解的葡聚糖链为水解酶如β-淀粉酶和异淀粉酶提供合适的底物，磷酸酶SEX 4和LSF 2的协调作用是必需的。迄今为止，很少有人知道这些过程的调节在淀粉颗粒表面。同时，还不清楚表面结构/性质如何影响淀粉代谢中涉及的酶，反之亦然，颗粒的这些表面性质/形态如何通过特定蛋白质/酶的作用详细改变。对于最近鉴定的蛋白ESV 1，这是必不可少的淀粉降解速率在黑暗阶段，它是在体外研究中显示，它改变二激酶介导的磷酸盐掺入。类似地，这种效应也发生在同源物LESV上。结果发现，这些影响不是由蛋白质-蛋白质相互作用引起的，而是由ESV 1和LESV在淀粉颗粒表面的特定结构处的结合引起的。结果表明，这两种蛋白质的葡聚糖结合特异性不同，ESV 1优先结合支链淀粉，LESV优先结合直链淀粉。ESV 1和LESV对其他淀粉代谢相关酶如BAM、伊萨、SEX 4、PHS 1和SS 1的影响也得到了证实。然而，确切的分子背景尚不清楚。在本提案中，将使用不同的实验方法并考虑到以下主要目标，对这一机制进行详细分析。(1)确定蛋白ESV 1和LESV结合的葡聚糖结构，以及哪个蛋白区域负责观察到的对淀粉代谢相关酶的影响。(2)分析淀粉代谢中受ESV 1和LESV影响的其他酶/蛋白质。(3)研究不同物种GWD和PWD的磷酸化模式以及ESV 1和LESV对这种模式的影响。(4)分析了不同突变体淀粉粒在日变化和老化过程中的表面结构特征，以及这些结构特征如何决定淀粉粒的大小、大小分布和形态特征。