Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves.: The phosphoglucan, water dikinase
Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves.: The phosphoglucan, water dikinase
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DOI：
10.1104/pp.104.055954
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发表时间：
2005-01-01
期刊：
影响因子：
7.4
通讯作者：
Ritte, G
中科院分区：
文献类型：
--
作者：
Kötting, O;Pusch, K;Ritte, G
通过葡聚糖水二激酶（GWD; EC 2.7.9.4）磷酸化支链淀粉是淀粉代谢中的重要步骤。这是由GWD缺陷型植物的淀粉过量表型所指示的，例如拟南芥属（Arabidopsis thaliana）的sex 1 -3突变体。为了确定依赖于葡聚糖结合磷酸的淀粉相关酶，我们研究了从拟南芥野生型叶片中提取的蛋白质与磷酸化或非磷酸化淀粉颗粒的结合。使用重组马铃薯（Solanum tuberosum）GWD在体外预磷酸化从sex 1 -3突变体制备的颗粒。作为对照，使用未改性的不含磷酸盐的颗粒。一种尚未鉴定的蛋白质被鉴定为优先与磷酸化淀粉结合。该蛋白的C-末端部分与GWD的C-末端部分具有相似性。这种新的蛋白质磷酸化淀粉颗粒，但只有在预磷酸化与GWD。该酶将ATP的β-P转移到磷酸葡聚糖，而γ-P以正磷酸盐的形式释放。因此，新的蛋白质被命名为磷酸葡聚糖，水双激酶（PWD）。与优先磷酸化葡萄糖单元的C6位置的GWD不同，PWD主要（或排他地）磷酸化C3位置。拟南芥叶片原生质体和叶绿体组分的Western印迹分析揭示了PWD的质体定位。PWD与淀粉颗粒的结合在净淀粉分解过程中强烈增加。通过RNAi或T-DNA插入降低PWD表达的转基因拟南芥植物表现出淀粉过量表型。因此，在拟南芥叶片中，淀粉周转需要PWD和GWD的密切合作。
The phosphorylation of amylopectin by the glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step within starch metabolism. This is indicated by the starch excess phenotype of GWD-deficient plants, such as the sex1-3 mutant of Arabidopsis (Arabidopsis thaliana). To identify starch-related enzymes that rely on glucan-bound phosphate, we studied the binding of proteins extracted from Arabidopsis wild-type leaves to either phosphorylated or nonphosphorylated starch granules. Granules prepared from the sex1-3 mutant were prephosphorylated in vitro using recombinant potato (Solanum tuberosum) GWD. As a control, the unmodified, phosphate free granules were used. An as-yet uncharacterized protein was identified that preferentially binds to the phosphorylated starch. The C-terminal part of this protein exhibits similarity to that of GWD. The novel protein phosphorylates starch granules, but only following prephosphorylation with GWD. The enzyme transfers the beta-P of ATP to the phosphoglucan, whereas the gamma-P is released as orthophosphate. Therefore, the novel protein is designated as phosphoglucan, water dikinase (PWD). Unlike GWD that phosphorylates preferentially the C6 position of the glucose units, PWD phosphorylates predominantly (or exclusively) the C3 position. Western-blot analysis of protoplast and chloroplast fractions from Arabidopsis leaves reveals a plastidic location of PWD. Binding of PWD to starch granules strongly increases during net starch breakdown. Transgenic Arabidopsis plants in which the expression of PWD was reduced by either RNAi or a T-DNA insertion exhibit a starch excess phenotype. Thus, in Arabidopsis leaves starch turnover requires a close collaboration of PWD and GWD.