Structural biology of C4 enzymes related in regulation of phosphoenolpyruvate carboxylase

关于CO_2同化，植物分为C3植物和C4植物两大类。C3植物的光呼吸途径是由核酮糖1，5-二磷酸羧化酶/加氧酶的氧化反应引起的，在水分胁迫和高温等环境条件下，C3植物的光呼吸速率增加，导致CO2同化效率降低。相比之下，C4植物有一条独特的大气CO_2参与PEPC的途径。PEPC对相对惰性的碳酸氢根离子有很高的亲和力。与C_3植物相比，C_4形式的PEPC在C_4植物中大量表达，并在C_4光合作用中催化大气CO_2固定的第一步。近年来，通过重组DNA技术将C_4特异基因导入C_3植物以提高C_3光合作用中CO_2固定效率的尝试已经展开。然而，要提高效率…仍然是困难的。由于PEPC的活性受代谢产物如苹果酸和葡萄糖6-磷酸的调节，并通过可逆的磷酸化进行共价修饰，因此PEPC的活性更高。因此，研究PEPC的分子机制以及调控PEPC的相关酶对植物的改良具有重要意义。本课题对PEPC突变体、PEPC特异性激酶PEPC-PK和C4型NADP依赖的苹果酸酶(NADP-ME)进行了结晶学和生化研究。首先，用X射线结晶学方法确定了对负变构效应不敏感的K620S突变体的结构。该结构揭示了PEPC的钝化机理。其次，位于二聚体界面边界的葡萄糖6-磷酸结合位点已经通过使用三维结构信息的定点突变来确定(J.Biol.Chem.280,11798-11806(2005年))。这项研究既提供了积极效应器是如何绑定的，也提供了PEPC是如何由效应器调节的。第三，对NADP-ME进行了提纯和结晶。C4特定的酶产生苹果酸，它被称为PEPC的负变构效应。因此，其分子机制有望为C4光合作用的调控提供新的思路。得到的晶体很薄很小，但在Spring-8中它的衍射率高达3埃分辨率。目前，与几种底物类似物的共结晶和结构测定正在进行中。第四，对PEPC-PK进行了纯化和结晶。据认为，这种特定的激酶作用于PEPC的特定位置会改变其构象，从而转化为具有酶活性的PEPC。高纯度的酶已结晶，但其体积太小，无法进行X射线衍射法测定。目前，PEPC-PK的结晶主要是通过多种沉淀剂和盐等进行的。最后介绍了PEPC的调控和羧化的分子机制(植物生物学年刊55，69-84(2004)，Arch.BioChem.BiPhys.414,170-179(2003))。较少

With respect to CO_2 assimilation, plants are divided into two types called C3 plants and C4 plants. The photorespiration pathway in C3 plants is caused by the oxygenation reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase and the rate of photorespiration in C3 plants is increased under the environment conditions such as water stress and high temperature, which leads to decrease the efficiency of CO_2 assimilation. In contrast, C_4 plants have a unique pathway of atmospheric CO_2 involved in PEPC. PEPC has a high affinity for the relatively inert bicarbonate ion. C_4-form PEPC is abundantly expressed in C_4 plants compared to that in C_3 plants and catalyzes the first committed step for the fixation of atmospheric CO_2 during C_4-photosynthesis. Recently, some trials to introduce C_4 specific genes into C_3 plants have been made to improve the efficiency of CO_2 fixation in C_3 photosynthesis though recombinant DNA techniques. However, it is still difficult to improve the effi … More ciency because the activities of PEPC are regulated by metabolites such as malate and glucose 6-phosphate and covalent modification by reversible phosphorylation. Thus, the molecular mechanism of PEPC and enzymes related in regulation of PEPC are crucial in improvement of the plants.In this project, the crystallogphic and biochemical studies of PEPC mutant, PEPC specific kinase called PEPC-PK and C4 type NADP dependent malic enzyme(NADP-ME) were performed. First of all, the structure of K620S mutant, which is insensitive to the negative allosteric effector, was solved by X-ray crystallography. The structure revealed the insensitive mechanism of PEPC. Secondary, the glucose 6-phosphate binding site, which is located at the boundary of the dimer interface, has been determined by site-directed mutagenesis using three dimensional structure information (J.Biol.Chem.280,11798-11806(2005).). This study provided both how the positive effector is bound and how PEPC is regulated by the effector. Thirdly, the purification and crystallization of NADP-ME have been done. The C4 specific enzyme produces the malate which is known as the negative allosteric effector of PEPC. Therefore, the molecular mechanism is expected to provide the insight of C4 photosynthesis regulation. The obtained crystal was very thin and small, but it diffracted up to 3 angstrom resolution in SPring-8. The co-crystallization with several kinds of substrate analogue and structural determination are currently in progress. Fourthly the purification and crystallization of PEPC-PK have been performed. The phosphrylatioin of specific site of PEPC by this specific kinase is believed to change the conformation, which convert into enzymatically active PEPC. The highly purified enzyme was crystallized but it is too small for X-ray diffraction measurement. At present the crystallization of PEPC-PK is performed by several kinds of precipitants and salts and so on. At last, the two review papers, which mentioned the molecular mechanisms of regulation and carboxylation of PEPC, are reported (Annu.Rev.Plant Biol.55,69-84(2004)., Arch.Biochem.Biophys., 414,170-179(2003). Less