Structural biology of C4 enzymes related in regulation of phosphoenolpyruvate carboxylase

就CO_2同化而言，植物可分为C3植物和C4植物。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。而C_4植物有一条独特的CO_2释放途径参与PEPC。PEPC对相对惰性的碳酸氢根离子具有高亲和力。C_4型PEPC在C_4植物中的表达量高于C_3植物，是C_4光合作用中固定大气CO_2的第一个关键步骤。近年来，利用DNA重组技术将C_4特异基因导入C_3植物，以提高C_3植物光合作用中的CO_2固定效率。然而，提高其效率仍然是困难的。 ...更多信息 这是因为PEPC的活性受代谢产物如苹果酸和葡萄糖6-磷酸的调节，并通过可逆磷酸化进行共价修饰。本研究对PEPC突变体、PEPC特异性激酶PEPC-PK和C4型NADP依赖性苹果酸酶（NADP-ME）进行了晶体学和生物化学研究。首先，利用X射线晶体学方法解析了对负变构效应不敏感的K620 S突变体的结构。结构揭示了PEPC的钝感机理。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诱变来确定（J.Biol.Chem.280，11798 -11806（2005）.）。本研究提供了阳性效应子如何结合以及效应子如何调节PEPC。第三，对NADP-ME进行了纯化和结晶。C4特异性酶产生苹果酸，其被称为PEPC的负变构效应物。因此，C4光合作用调控的分子机制有望为C4光合作用调控提供新的思路。所获得的晶体非常薄且小，但其在SPring-8中衍射高达3埃分辨率。目前正在进行与几种底物类似物的共结晶和结构测定。第四，对PEPC-PK进行了纯化和结晶。这种特异性的磷酸化酶可以磷酸化PEPC的特定位点，从而改变PEPC的构象，使其转化为具有酶活性的PEPC。高纯度的酶结晶，但它太小的X-射线衍射测量。目前，PEPC-PK的结晶是通过几种沉淀剂和盐等进行的。最后，报道了两篇综述文章，其中提到了PEPC的调节和羧化的分子机制（Annu.Rev.PlantBiol.55，69 -84（2004），Arch. Biochem. Bioprotein.，414，170 -179（2003）。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