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

该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通过普遍存在的“可逆蛋白质磷酸化”过程进行调节的兴趣。整体调查处理具体与磷酸烯醇式丙酮酸羧化酶（PEPC）和蔗糖合成酶（SuSy），两个“目标”酶进行磷酸化在光合作用的叶子和/或固氮根瘤的大豆，豌豆，和其他豆类。虽然PEPC最为人所知的是其在高粱、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的主要作用，但它也在各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色藻类的碳/氮代谢。植物PEPC的这种“多面性”性质使其成为继续研究其（a）由“PEPC-激酶”（PpcK）（磷酸化/活化PEPC的新型高度调节的蛋白激酶）的复杂控制和（B）新兴的生物化学结构-功能关系的极具吸引力的主题。沿着这些路线，该更新项目的三个具体实验目标中的两个侧重于植物PpcK以及高粱叶和绿藻PEPC的分子/生物化学分析。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“库”组织中的主要蔗糖代谢酶，包括豆科植物根瘤、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主要源于这样一个事实，即与PEPC一样，它是少数几种磷酸化植物酶之一，具有在植物和体外鉴定的特定“靶”氨基酸残基。此外，这种“靶”酶具有被植物蛋白激酶的至少两个不同亚家族在多个位点磷酸化的记录潜力。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。这些不确定性将在本项目期间通过生物化学、免疫学和质谱方法相结合的实验来解决。这一多管齐下的基础研究和发现项目的意义在于：（a）加强对参与植物初级代谢的两种重要酶的复杂调控和结构-功能关系的基本理解，和（B）提供对唯一“靶向”其中一种的独特蛋白激酶的详细分子见解。因此，从这项研究的结果将有助于显着的必要的基础建设性操纵植物碳/氮代谢及其控制机制，在叶片和豆科植物根瘤的生物技术方法。

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.