Structure and Function Diversity of Phosphotransfer Proteins and their Sequence Family Relatives

该项目的目标是深入了解磷酸转移反应在生物学中的关键作用，并探索结构家族中蛋白质的活动范围。除了其功能已知的蛋白质之外，酶家族成员还包括从基因组测序计划中出现的蛋白质，迄今为止可用的结构/序列信息表明它们在序列数据库中被错误地注释。所获得的信息将被用于将活性位点结构与催化作用联系起来，并识别标记物，这些标记物可以应用于研究的每个家族的其他蛋白质的功能分配。这些研究将导致新的生物化学和途径的发现，并将提供从祖先活性位点模板的功能进化的见解。两种蛋白质提供了用于选择序列家族的框架：（1）丙酮酸磷酸二激酶（PPDK），一种催化ATP、Pi和丙酮酸与AMP、PPi和磷酸烯醇丙酮酸（PEP）的相互转化的多结构域酶。(2)PEP β，一种催化PEP重排为膦酰丙酮酸的酶，该反应是所有天然膦酸盐合成的主要步骤。这是一个结构/功能的合作努力，重点是通过X射线晶体学获得的结构数据。X射线结构提供了理解酶机制的基础，并通过定点诱变和抑制剂设计测试建议。除上述蛋白质外，将研究的新蛋白质包括（1）来自M.结核病，Rv 1127 c，缺乏丙酮酸结合域，因此有望导致一种新的磷酸转移途径的发现;（2）PEP β/异柠檬酸裂解酶家族的成员，特别强调与康乃馨花瓣死亡相关的蛋白质，在序列数据库中被注释为β，但实际上是一种新的裂解酶。该项目将为各级学生提供研究机会：博士后研究员，研究生和本科生。它将导致发现新的蛋白质功能和新的生物学途径。这些结果将为生物技术开发提供新的目标，并将通过在科学期刊上发表，在蛋白质数据库中存放坐标以及在序列数据库中更正注释来传播。

The goal of this project is to gain insight into the pivotal role that phosphotransfer reactions play in biology, and to explore the range of activities performed by proteins in a structural family. In addition to proteins whose function are already known, the enzyme family members include proteins emerging from genome sequencing projects, that the structural/sequence information available so far indicates that they are incorrectly annotated in sequence databases. The information gained will be used to relate active site structure to catalysis, and to identify markers, which can be applied in the assignment of function to other proteins from each family studied. These studies will lead to the discovery of novel biochemistry and pathways, and will provide insights into the evolution of function from ancestral active site templates. Two proteins provide the framework for selection of sequence families: (1) Pyruvate phosphate dikinase (PPDK), a multi-domain enzyme that catalyzes the inter-conversion of ATP, Pi and pyruvate with AMP, PPi and phosphoenolpyruvate (PEP). (2) PEP mutase, an enzyme that catalyzes the rearrangement of PEP to phosphonopyruvate, a reaction that serves as the major entry step into the synthesis of all natural phosphonates. This is a structure/function collaborative effort, focused on structural data that are obtained by x-ray crystallography. The x-ray structures provide the basis for understanding enzyme mechanisms, and for testing proposals by site-directed mutagenesis and inhibitor design. In addition to the above proteins, new proteins that will be investigated include (1) a PPDK homologue from M. tuberculosis, Rv1127c, lacking the pyruvate binding-domain, and therefore expected to lead to the discovery of a novel phosphotransfer pathway; (2) members of the PEP mutase/isocitrate lyase enzyme family, with special emphasis on a protein associated with carnation flower petal death, which is annotated as a mutase in sequence databases, but is actually a novel lyase.Broader Impact: The project will provide research opportunities to students at all levels: post-doctoral fellows, graduate students, and undergraduate students. It will lead to the discovery of novel protein functions and novel biological pathways. The results will provide new targets for biotechnological exploitation, and will be disseminated by publication in scientific journals, by depositing the coordinates in the Protein Data Bank, and by correcting annotations in sequence databases.