Structural biology of C4 enzymes related in regulation of phosphoenolpyruvate carboxylase

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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