Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Biomedical Biological Sciences Research Building, 741 South Limestone, Lexington, KY 40536-0509, USA.
Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15379-84. doi: 10.1073/pnas.1009386107. Epub 2010 Aug 2.
Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.
生物利用碳水化合物作为重要的储能分子。淀粉是植物中主要的碳水化合物储存分子，而糖原则在动物中被利用。淀粉是一种不溶于水的聚合物，需要激酶和磷酸酶的协同作用来溶解葡聚糖的外表面，并介导淀粉的分解代谢。所有已知的植物基因组都编码葡聚糖磷酸酶淀粉过量 4 型（SEX4）。SEX4 可以去磷酸化淀粉颗粒表面和可溶性磷酸葡聚糖，是进行性淀粉代谢所必需的。SEX4 作为葡聚糖磷酸酶的功能的物理基础目前尚不清楚。本文报道了 SEX4 的晶体结构，包含磷酸酶、碳水化合物结合和 C 末端结构域。SEX4 的三个结构域折叠成一个紧凑的结构，具有广泛的结构域间相互作用。SEX4 的 C 末端结构域完整地折叠到磷酸酶结构域的核心，并对其稳定性至关重要。磷酸酶和碳水化合物结合结构域直接相互作用，并将磷酸酶活性位点定位到碳水化合物结合位点的单一连续口袋中。磷酸酶结构域残基 F167 的突变，该残基形成口袋的底部并桥接两个结构域，选择性地影响 SEX4 作为葡聚糖磷酸酶的功能。这些结果共同揭示了 SEX4 的独特三级结构，为其作为葡聚糖磷酸酶的功能提供了物理基础。
