集胞藻镁原卟啉IX甲基转移酶的分子机理研究

Mg-protoporphyrin IX methyltransferase (MgPMT) is an enzyme in chlorophyll biosynthesis, which catalyzes the methylation of Mg-protoporphyrin IX to form Mg-protoporphyrin IX monomethyl ester. It belongs to the S-adenosylmethionine-dependent methyltransferase family. It is believed that both the substrate and the product of this enzyme are implicated in signalling between the plastid and nucleus. The activity of this enzyme and the interaction with the subunit ChlH of Mg chelatase are important in chlorophyll biosynthesis regulation. So far, the substrate recognizing and activity regulation of this enzyme is needed to discover. The structural information about this enzyme is still lacking in PDB. Here we propose to use structural and biochemical analysis as primary tools, including enzyme kinetics, to determine the high-resolution structure and biochemical activity of MgPMT from Synechocystis sp. Combining the structural and biochemical data and mutant analysis, we intend to discover the catalytic mechanism of MgPMT. This work will provide direct proof and molecular basis for our understanding of the role of MgPMT in chlorophyll biosynthesis.

镁原卟啉IX甲基转移酶(MgPMT)是叶绿素生物合成途径中的酶，催化镁原卟啉IX形成镁原卟啉IX单甲基酯，属于S-腺苷-甲硫氨酸依赖的甲基转移酶家族。已有的研究表明，MgPMT的底物和产物参与了细胞中质体－核的信号转导，该酶与镁螯合酶催化亚基ChlH的相互作用及活性变化是叶绿素合成调控中的重要方式。但目前对MgPMT的底物识别机制和活性调控方式还缺乏足够了解，PDB中也缺乏相关的结构信息。本课题运用结构生物学和生物化学的方法，测定和分析集胞藻中MgPMT的精细结构、生化活性和酶动力学数据，了解其底物特异性和反应机制。结合生化酶学分析和结构生物学信息以及突变体验证，来阐明MgPMT的催化机制,从而为理解MgPMT在叶绿素合成中的酶学机制提供直接证据和分子生物学基础。

镁原卟啉IX 甲基转移酶是叶绿素生物合成途径中的酶（在光合生物中由ChlM 基因编码），催化镁原卟啉IX 形成镁原卟啉IX 单甲基酯，这是叶绿素分支途径的第二步反应。镁原卟啉IX 甲基转移酶属于S-腺苷-甲硫氨酸依赖的甲基转移酶家族。我们通过原核表达与纯化获得了来源于集胞藻（Synechocystis sp.）的纯度较高的镁原卟啉IX 甲基转移酶（SyChlM）蛋白，通过晶体筛选和优化获得了SyChlM、SyChlM-SAM和SyChlM-SAH的晶体，通过单波长反常散射法（SAD）解析了后两者1.6 Å和1.7 Å晶体结构。在此结构基础上，使用计算机模拟的方法将底物分子MgP模拟到SyChlM的结构当中。根据晶体结构和底物模型，我们描述了SAM/SAH和MgP的结合口袋。结合定点突变、酶活性测定和ITC等生物化学实验，我们找到了两个关键的催化氨基酸His139和Tyr28，讨论了它们在催化过程中的作用，并根据结构等信息提出了SyChlM的催化机制。