RUI: Uncovering the Occurrence and Mechanism of Regulatory Phosphorylation of Non Photosynthetic Pyruvate,Orthophospate Dikinase in C3 Plants

RUI：揭示C3植物中非光合丙酮酸、正磷酸二激酶调节性磷酸化的发生和机制

• 批准号: 0094497
• 负责人: Chris Chastain
• 金额: $ 7.44万
• 依托单位: Minnesota State University Moorhead
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-15 至 2003-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0094497&HistoricalAwards=false
• 关键词: RUI; Uncovering; Occurrence; Mechanism; Regulatory

The enzyme pyruvate,orthophosphate dikinase (PPDK) is most well known asan enzyme of the C4 photosynthetic pathway where it catalyzes theformation of PEP (phosphoenolpyruvate), the primary CO2 acceptormolecule, from pyruvate. Because it is a key enzyme in the C4 pathway,PPDK activity is highly regulated so the overall pathway can functionoptimally in CO2 assimilation. This regulation is conferred byreversible phosphorylation of a PPDK active-site threonine, with theenzyme being inactive in the phosphorylated state. A single bifunctionalprotein kinase/phosphatase enzyme, PPDK regulatory protein (RP),catalyzes this regulatory phosphorylation and dephosphorylation. PPDK isalso present in C3 plants although it does not participate inphotosynthesis. Further, it is present in all tissues of the plant butin very low amounts. Its function in C3 plants is essentially unknown,although it is proposed to participate in cellular metabolism andguard cell function. Recently, it was demonstrated that PPDK in leavesof C3 plants undergoes light/dark mediated regulatory phosphorylationsimilar to C4 leaf PPDK. The major aim of this project is to furthercharacterize C3 plant PPDK regulatory phosphorylation by identifyingorgan, tissue, cellular, and subcellular sites of regulatory PPDKphosphorylation. Further, the C3 RP or RP-like activity mediatingreversible phosphorylation of PPDK in C3 plant tissue will bescrutinized in vitro for understanding its biochemical nature. Finally,this project seeks to clone the gene for C4 PPDK RP from a maize cDNAlibrary. This will provide the "ultimate" tool for resolving theperplexing riddle of how this uniquely bifunctional enzyme is regulatedin C4 plants, as well as the requisite gene probe for understanding itsprecise expression in C3 and C4 plants. In summary, the experimentaloutcomes of this project will enable the hitherto unknown metabolicfunction(s) of PPDK in C3 plants to be elucidated.

丙酮酸正磷酸二激酶 (PPDK) 是最著名的 C4 光合作用酶，它催化丙酮酸形成 PEP（磷酸烯醇丙酮酸），这是主要的 CO2 受体分子。由于它是 C4 途径中的关键酶，PPDK 活性受到高度调节，因此整个途径可以在 CO2 同化中发挥最佳作用。这种调节是由 PPDK 活性位点苏氨酸的可逆磷酸化赋予的，酶在磷酸化状态下无活性。一种双功能蛋白激酶/磷酸酶，即 PPDK 调节蛋白 (RP)，可催化这种调节性磷酸化和去磷酸化。 PPDK 也存在于 C3 植物中，尽管它不参与光合作用。此外，它存在于植物的所有组织中，但含量非常低。尽管有人提出它参与细胞代谢和保护细胞功能，但它在 C3 植物中的功能基本上是未知的。最近，已证明 C3 植物叶子中的 PPDK 经历与 C4 叶子 PPDK 类似的光/暗介导的调节性磷酸化。 该项目的主要目的是通过鉴定调节性 PPDK 磷酸化的器官、组织、细胞和亚细胞位点来进一步表征 C3 植物 PPDK 调节性磷酸化。此外，将在体外检查介导C3植物组织中PPDK可逆磷酸化的C3 RP或RP样活性，以了解其生化性质。最后，该项目试图从玉米 cDNA 文库中克隆 C4 PPDK RP 基因。这将为解决这种独特的双功能酶在 C4 植物中如何调节这一令人困惑的谜题提供“终极”工具，并为了解其在 C3 和 C4 植物中的精确表达提供必要的基因探针。总之，该项目的实验结果将能够阐明 C3 植物中 PPDK 迄今为止未知的代谢功能。