植物镁离子螯合酶I与D亚基的结构与机理

Photosynthesis supplies life with energy, oxygen and organics through transforming solar energy into chemical energy. Chlorophyll molecules play a key role in photosynthesis, which mediates the absorption, transfer and conversion of solar energy. The biosynthesis of chlorophyll originates from the chlorophyll branch of the tetrapyrrole synthesis pathway in organism. Magnesium chelatase is the key enzyme in this branch and catalyzing the chelation of magnesium into the protoporphyrin IX to form magnesium protoporphyrin IX, which is essential for physiological function. Magnesium chelatase is composed of three subunits of I, D and H. The reaction process requires the participation of ATP. At present, although people have obtained genetic mutants of magnesium chelatase subunits of Arabidopsis and other plants, and carried out research on their respective functions, little is known about the structures of the subunits, the holoenzyme complex, the assembly mechanism and regulatory mechanism of catalysis. Using protein crystallography combined with cryo-electron microscopy techniques and biochemical methods, this project aims to determine the structures of magnesium chelatase subunit I, D and I-D complex in different conditions. This will reveal the assembly mechanism of subunits I and D, and disclose the underlying functional mechanism of I-D subcomplex in holoenzyme assembly and function.

光合作用将太阳能转化为化学能，为自然界绝大多数生命活动提供能量、氧气和有机物。叶绿素分子在光合作用中发挥着关键作用，介导了太阳光能的吸收、传递和转换。叶绿素的生物合成源于有机体四吡咯合成途径中的叶绿素分支。镁离子螯合酶是这一分支的关键酶，负责将原卟啉IX加镁生成镁原卟啉IX，起始叶绿素合成，生理功能尤为重要。镁离子螯合酶全酶由I、D和H三种亚基组成，反应过程需要ATP的参与。目前，虽然人们已经获得了拟南芥等植物镁离子螯合酶各个亚基的遗传学突变体，并对各自的功能进行了一些研究，但是对于其各亚基及全酶复合体的结构、组装机制、以及催化和调控机理并不了解。本项目拟利用蛋白质晶体学，结合冷冻电镜技术和生物化学方法，解析镁离子螯合酶I、D亚基及I-D复合体在不同状态下的结构。揭示I与D亚基形成寡聚复合物的组装机制，阐明I-D亚基复合体在镁离子螯合酶全酶组装中发挥功能的分子机理。

本项目主要围绕植物叶绿素合成过程中的关键酶-镁离子螯合酶I与D亚基的结构与机理开展研究。项目实施四年，除完成既定的研究任务和目标外，也开展并完成发表了其它植物重要代谢酶的研究工作，取得了多项科研成果，包括：①对镁离子螯合酶I、D亚基及I-D复合体进行了详细的蛋白表达纯化、蛋白状态测定、酶活性分析、蛋白结晶及晶体优化、负染及冷冻电镜样品的制备和检测等实验研究，解析了I亚基的晶体和电镜结构，收集了D亚基的300kV冷冻电镜数据，分析发现了I亚基与ATP的结合口袋及与催化ATP水解相关的多个关键氨基酸残基，揭示了I和D亚基在负染和冷冻电镜下均呈现环状寡聚体的状态。②揭示了真核生物磷脂酶D水解磷脂产生磷脂酸的分子机制，挖掘并阐明了蕨类植物新型查尔酮合酶的功能和催化机制，揭示了两种BAHD酰基转移酶的催化机制等，论文发表于Cell Research、Frontiers in Plant Science（两篇）和 Journal of Integrative Plant Biology。