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

这个基础研究项目的重点是这个实验室长期以来对植物代谢酶的兴趣，以及通过普遍存在的“可逆蛋白质磷酸化”过程对它们的调节。整个研究特别涉及磷酸烯醇式丙酮酸羧化酶(PEPC)和蔗糖合成酶(SUSY)，这两种“靶标”酶在光合作用的叶片和/或大豆、豌豆和其他豆类的固氮根瘤中经历磷酸化。虽然PEPC因其在高粱、玉米、甘蔗和菠萝等作物光合作用过程中最初固定大气二氧化碳的关键作用而闻名，但它也在一系列不同的非光合作用环境中发挥作用，例如豆类根瘤和单细胞绿藻的碳/氮代谢。植物PEPC的这种“多方面”性质使其成为一个极具吸引力的继续研究的主题：(A)“PEPC-激酶”(PpcK)的复杂控制，PpcK是一种新型的、高度调控的蛋白激酶，可使PEPC磷酸化/激活，以及(B)新出现的生化结构-功能关系。沿着这些思路，这个更新项目的三个具体实验目标中的两个专注于植物PPCK的分子/生化分析，以及高粱叶和绿藻的PEPC。这项研究的第三个也是最后一个焦点是Susy，它可能是植物中各种非光合作用“库”组织中的主要蔗糖代谢酶，包括豆类根瘤、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在很大程度上源于这样一个事实，即与PEPC一样，它是植物体内和体外发现的少数几种具有特定“靶标”氨基酸残基的磷酸化植物酶之一(S)。此外，这种“靶标”酶有可能被至少两个不同的植物蛋白激酶亚家族在多个位置上的磷酸化。然而，这种磷酸化事件(S)的确切作用及其在植物中的动态可逆性尚未确定。这个多管齐下的基础研究和发现项目的意义在于，它将(A)加强对植物初级代谢中两种重要酶的基本了解，因为它们的复杂调节和结构-功能关系，以及(B)提供对独特的蛋白激酶的详细的分子洞察。因此，这项研究的结果将为利用生物技术方法建设性地操纵植物的碳/氮代谢及其在叶和豆科植物根瘤中的调控机制提供必要的基础。

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.