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Abstract：
Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli (EcAGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a \"locked\" to a \"free\" state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of EcAGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of EcAGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.
摘要：
糖原和淀粉是自然界中主要的碳和能量储备多糖，为生物体提供生存优势。负责这种多糖的生物合成和降解的酶机制的演变，领导了控制装配和拆卸率的机制的发展，根据细胞能量需求储存和回收葡萄糖。四聚体酶ADP-葡萄糖焦磷酸化酶（AGPase）催化并调节两种α-聚葡聚糖生物合成的初始步骤。AGPase通过从细胞能量通量中感知代谢物而表现出协同性和变构调节。对AGPase中变构信号转导机制的理解是一项长期挑战。在这项工作中,我们公开了来自大肠杆菌的范式同四聚体AGPase（EcAGPase）的冷冻EM结构，与正或负生理变构调节剂复合，1,6-二磷酸果糖(FBP)和AMP,两者的分辨率均为3.0。引人注目的是，结构显示FBP与变构裂隙深度结合并与AMP位点重叠。因此,FBP促进调节环的一致构象转换，RL2，从“锁定”状态到“空闲”状态，调节ATP结合和激活酶。我们的生物物理和生物信息学证据强烈支持这一观点，并仔细分析了EcAGPase单点突变体的大量酶动力学数据。冷冻EM结构揭示了酶的变构和催化组分之间的残基相互作用网络（RIN），提供有关如何通过四聚体传输信令信息的唯一详细信息，从中产生合作。总之,通过冷冻EM可视化的构象状态揭示了EcAGPase的调节机制，奠定了基础，从分子水平上了解细菌糖原生物合成的变构控制。
