RUI: Pyruvate,Phosphate Dikinase Regulatory Protein in C3 Plants

在植物中催化代谢反应的酶受到高度调节，以防止生物合成和能量生产中的有害低效。植物细胞用来调节代谢酶活性的最普遍的机制之一是磷酸分子与酶蛋白质结构内的氨基酸的酶催化键合。这种形式的酶调节，称为可逆蛋白磷酸化，是控制细胞中数千种酶的机制。植物代谢酶丙酮酸，磷酸二激酶（PPDK），本研究的主题，是一个可逆的磷酸化调节酶的例子。PPDK催化磷酸烯醇丙酮酸的合成，磷酸烯醇丙酮酸是氨基酸和细胞壁聚合物等多种分子的“构建单元”。赋予PPDK调节的是酶RP（PPDK调节蛋白）。RP是该研究项目的主要焦点，以前的研究已经证明它具有前所未有的独一无二的催化性能。然而，这些特性的结构基础仍然是一个谜，因为RP基因一直拒绝克隆，直到今年PI和同事从拟南芥中分离出该基因。他们对编码多肽的分析表明，RP代表了一种全新的磷酸化调节酶，这种酶迄今为止在植物或动物中是未知的。本研究的目的是利用RP基因的克隆，阐明酶的独特催化性能的结构基础。在这样做的时候，很少了解的机制，支配RP的PPDK的调节也将被揭示。这些目标将通过确定拟南芥RP蛋白的三维结构来实现。分子和遗传学工具将进一步确定拟南芥基因组中两个不同的RP基因在植物中的空间表达。最后，结合生物化学和分子遗传学的方法将阐明这两种RP酶之间的功能差异。更广泛的影响包括：（一）增加我们在植物碳代谢方面的知识基础，以开发纤维素乙醇生物燃料;（二）招募和培训本科生，使其成为我国新兴生物技术部门的未来科学家。

Enzymes that catalyze metabolic reactions in plants are highly regulated in order to prevent detrimental inefficiencies in biosynthesis and energy production. One of the most prevalent mechanisms plant cells use to regulate the activity of metabolic enzymes is enzyme-catalyzed bonding of a phosphate molecule to an amino acid within the enzyme's protein structure. This form of enzyme regulation, termed reversible protein-phosphorylation is the mechanism controlling thousands of enzymes in the cell. The plant metabolic enzyme pyruvate,phosphate dikinase (PPDK), the subject of this research, is an example of an enzyme regulated by reversible phosphorylation. PPDK catalyzes the synthesis phosphoenolpyruvate, a "building block" for a diverse set of molecules such as amino acids and cell-wall polymers. Conferring regulation of PPDK is the enzyme RP (PPDK Regulatory Protein). RP, the primary focus of this research project, had been demonstrated by previous studies to have unprecedented, one-of-a-kind catalytic properties. However, the structural basis for these properties has remained a mystery as the gene for RP had resisted cloning until this year, when the PI and coworkers isolated the gene from Arabidopsis. Their analysis of the encoded polypeptide showed that RP represents a fundamentally new kind of phosphorylating-regulatory enzyme hitherto unknown in plants or animals. This research aims to capitalize on the cloning of the RP gene by elucidating the structural basis of the enzyme's unique catalytic properties. In so doing, the poorly understood mechanism that governs RP's regulation of PPDK will also be revealed. These aims will be achieved by determining the three-dimensional structure of the Arabidopsis RP protein. Molecular and genetic tools will further establish how the two distinct RP genes represented in the Arabidopsis genome are spatially expressed in planta. Finally, a combined biochemical and molecular-genetic approach will elucidate the functional differences between these two RP enzymes. Broader impacts include (i) increasing our knowledge base in plant carbon metabolism for developing cellulosic ethanol biofuels, and (ii) the recruitment and training of undergraduate students as future scientists for our Nation's bourgeoning biotechnology sector.