Synthesis of Glycogen in Bacteria and Starch in Plants: Evolution and Mechanism of Allosteric Control

ADP-葡萄糖焦磷酸化酶是植物中淀粉合成和细菌及其它生物中糖原合成的核心，作为碳储存聚合物。它是一个常见的生物技术目标，了解其调节对许多生物体中的淀粉/糖原合成有影响。 ADP-葡萄糖焦磷酸化酶活性的变构控制是一种重要但知之甚少的调节机制。 目前还不清楚这种酶是如何变构激活的，以及这种激活信号是如何在酶内结构性地传递的。该项目的目标是进行计算，结构和生物化学研究，以了解和表征细菌中的别构信号和别构效应物对ADP-葡萄糖焦磷酸化酶活性的相互作用，作为植物的模型。 PI预测了作为开关工作的共同结构元件的存在，但也预测了替代变构信号结合位点的存在。该项目还将通过培训本科生和研究生来促进教育，建立国际合作，并通过支持McNair-SROP等项目来促进代表性不足的少数民族参与研究。 该项目还将支持蛋白质科学的国际课程，其中包括研究的元素，为本科生和研究生提供独特的培训机会。 植物淀粉和细菌糖原生物合成途径提供了ADP-葡萄糖焦磷酸化酶的结构和变构调节功能的进化模型。尽管这些途径的重要性，控制这种酶的活性的变构机制在很大程度上是未知的。该项目包括计算，结构和生物化学研究，以了解和表征ADP-葡萄糖焦磷酸化酶家族中变构信号的传递和变构效应物的相互作用。 据预测，有一个共同的结构元件，作为开关和一个替代的变构信号引导的第二个网站。本论文的主要研究目标是：1）从系统发育树的不同分支中筛选出具有双重变构机制的酶; 2）获得变构突变体的晶体结构（“关闭”），以检验某些残基在ADP-葡萄糖焦磷酸化酶中形成保守的变构开关的假设;和3）进行计算研究（分子动力学、相关运动分析和网络途径分析）以预测从每个推定的变构位点激活的信号传导途径。

ADP-glucose pyrophosphorylase is central to starch synthesis in plants and glycogen synthesis in bacteria and other organisms, serving as carbon storage polymers. It is a common biotechnology target and an informed understanding of its regulation has implications for starch/glycogen synthesis in a host of organisms. Allosteric control of ADP-glucose pyrophosphorylase activity is an important but poorly understood regulatory mechanism. Presently it is unclear how the enzyme is allosterically activated and how that activation signal is transmitted structurally within the enzyme. The goal of this project is to perform computational, structural and biochemical studies to understand and characterize the allosteric signals and the interaction of the allosteric effectors on ADP-glucose pyrophosphorylase activity in bacteria as a model for plants. The PIs predict the presence of a common structural element that works as a switch but also the presence of an alternative allosteric signal binding site. This project will also contribute to education by training undergraduate and graduate students, establish international collaborations, and foster participation of underrepresented minorities in research by supporting programs such as McNair-SROP. This project will also support an international course on protein science that includes elements of the research, providing a unique training opportunity for undergraduate and graduate students. Plant starch and bacterial glycogen biosynthetic pathways provide models for the structure and evolution of allosteric regulatory functions of the enzyme ADP-glucose pyrophosphorylase. Despite the importance of these pathways, the allosteric mechanism that controls the activity of this enzyme is largely unknown. The project comprises computational, structural, and biochemical studies to understand and characterize the transmission of the allosteric signal and the interaction of the allosteric effectors in the ADP-glucose pyrophosphorylase family. It is predicted there is both a common structural element that works as a switch and an alternative allosteric signal guided by a secondary site. Three main research objectives will be pursued: 1) screen several enzyme forms from diverse branches of the phylogenetic tree for the presence of a dual allosteric mechanism; 2) obtain crystal structures of allosteric mutants ("switched off") in several different conditions with different ligands to test the hypothesis that certain residues form a conserved allosteric switch in ADP-glucose pyrophosphorylases; and 3) perform computational studies (molecular dynamics, correlated movement analysis, and network pathway analysis) to predict the signaling pathways of activation from each putative allosteric site.