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

这个项目的目标是深入了解磷转移反应在生物学中所起的关键作用，并探索蛋白质在结构家族中执行的活动范围。除了功能已知的蛋白质外，酶家族成员还包括从基因组测序项目中出现的蛋白质，到目前为止获得的结构/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来将活性部位结构与催化联系起来，并识别标记，这些标记可用于将功能分配给每个研究家族的其他蛋白质。这些研究将导致新的生物化学和途径的发现，并将为从祖先活性部位模板进化功能提供洞察力。有两个蛋白质为序列家族的选择提供了框架：(1)丙酮酸磷酸二激酶(PPDK)，它是一种多结构域的酶，可以催化ATP、PI和丙酮酸与AMP、PPI和磷酸烯醇式丙酮酸(PEP)的相互转化。(2)PEP变位酶，一种催化PEP重排为磷酸丙酮酸的酶，该反应是合成所有天然磷酸盐的主要步骤。这是一项结构/功能合作努力，重点放在通过X射线结晶学获得的结构数据上。X射线结构为了解酶的作用机制，以及通过定点突变和抑制剂设计来测试建议提供了基础。除上述蛋白质外，还将研究的新蛋白质包括：(1)结核分枝杆菌的PPDK同源物Rv1127c，它缺乏丙酮酸结合结构域，因此有望导致发现一种新的磷酸转移途径；(2)PEP变位酶/异柠檬酸裂解酶家族的成员，尤其侧重于与香石竹花瓣死亡相关的蛋白质，在序列数据库中被注释为变位酶，但实际上是一种新的裂解酶。广泛的影响：该项目将为各级学生：博士后、研究生和本科生提供研究机会。这将导致新的蛋白质功能和新的生物途径的发现。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、将坐标存放在蛋白质数据库中以及通过更正序列数据库中的注释来传播。

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.