Molecular/Biochemical Investigations of PEPC (and its Novel Ser/Thr-Kinase) and SuSy (Nodulin-100), Two Phosphorylated Metabolic Enzymes in Plants

本基础研究项目的重点是本实验室长期以来对植物代谢酶及其普遍存在的“可逆蛋白磷酸化”过程的调控的兴趣。整个研究特别涉及磷酸烯醇丙酮酸羧化酶（PEPC）和蔗糖合成酶（SuSy），这两种“靶”酶在大豆、豌豆和其他豆科植物的光合作用叶片和/或固氮根瘤中发生磷酸化。虽然PEPC最为人所知的是它在高粱、玉米、甘蔗和菠萝等作物光合作用期间对大气二氧化碳的初始固定中所起的主要作用，但它也在多种非光合作用环境中发挥作用，例如豆科植物根瘤和单细胞绿藻的碳/氮代谢。植物PEPC的“多面性”使其成为一个极具吸引力的主题，可以继续研究其(a)受“PEPC激酶”（PpcK）的复杂控制，PpcK是一种新型的，高度调节的蛋白激酶，可磷酸化/激活PEPC，以及(b)新兴的生化结构-功能关系。按照这些思路，这个更新项目的三个具体实验目标中的两个集中在植物PpcK和高粱叶片和绿藻PEPC的分子/生化分析上。本研究的第三个也是最后一个重点是SuSy，它可能是植物中各种非光合“汇”组织中的主要蔗糖代谢酶，包括豆科植物的根瘤、发育中的叶片、果实和种子以及块茎。对这种代谢酶的持续兴趣很大程度上源于这样一个事实，即像PEPC一样，它是在植物体内和体外都鉴定出的具有特定“目标”氨基酸残基的少数磷酸化植物酶之一。此外，这种“目标”酶具有被至少两个不同的植物蛋白激酶亚家族在多个位点磷酸化的潜力。然而，这种磷酸化事件的确切作用及其在植物中的动态可逆性都没有确定。在这个项目中，这些不确定性将通过生物化学、免疫学和质谱方法的结合来解决。这项多管齐下的基础研究和发现项目的意义在于，它将(a)从复杂的调控和结构功能关系方面增强对参与植物初级代谢的两种重要酶的基本理解，以及(b)提供对独特的蛋白激酶的详细分子洞察，该蛋白激酶专门“靶向”其中一种。因此，本研究的结果将为利用生物技术方法对植物叶片和豆科根瘤的碳/氮代谢及其控制机制进行建设性的操纵提供必要的基础。

This basic research project is focused on this laboratory's long-standing interest in plant metabolic enzymes and their regulation by the ubiquitous process of "reversible protein phosphorylation". The overall investigation deals specifically with phosphoenolpyruvate carboxylase (PEPC) and sucrose synthase (SuSy), two "target" enzymes that undergo phosphorylation in photosynthesizing leaves and/or nitrogen-fixing root nodules of soybeans, peas, and other legumes. While PEPC is best known for its cardinal role in the initial fixation of atmospheric carbon dioxide during photosynthesis by such crop plants as sorghum, corn, sugarcane and pineapple, it also functions in a diverse array of non-photosynthetic contexts, e.g., carbon/nitrogen-metabolism in legume root nodules and unicellular green algae. This "multifaceted" nature of plant PEPC makes it an extremely attractive subject for continued study of its (a) intricate control by "PEPC-kinase" (PpcK), the novel, highly regulated protein-kinase enzyme that phosphorylates/activates PEPC, and (b) emerging biochemical structure-function relationships. Along these lines, two of the three specific experimental objectives of this renewal project focus on the molecular/biochemical analysis of plant PpcK, and sorghum-leaf and green-algal PEPC. The third and final focus of this investigation is SuSy, likely the major sucrose-metabolizing enzyme in a variety of non-photosynthetic "sink" tissues in plants, including legume root nodules, developing leaves, fruits and seeds, and tubers. Continued interest in this metabolic enzyme largely stems from the fact that, like PEPC, it is one of but a handful of phosphorylated plant enzymes with a specific "target" amino-acid residue(s) identified both in planta and in vitro. In addition, this "target" enzyme has the documented potential for phosphorylation at multiple sites by at least two distinct subfamilies of plant protein kinases. However, neither the exact role of this phosphorylation event(s) nor its dynamic reversibility in planta is established. These uncertainties will be addressed experimentally during this project by a combination of biochemical, immunological and mass-spectroscopic approaches.The significance of this multi-pronged, basic research and discovery project is that it will (a) enhance the fundamental understanding of two important enzymes involved in plant primary metabolism in terms of their intricate regulation and structure-function relationships, and (b) provide detailed molecular insight into the unique protein kinase that "targets" one of them exclusively. As such, the results from this research will contribute significantly to the requisite underpinning for the constructive manipulation of plant carbon/nitrogen-metabolism and its control mechanisms in leaves and legume root nodules by biotechnological approaches.