Structure and function of PTP like inositol polyphosphatases

我的研究计划的总体目标是了解最近发现的与蛋白酪氨酸磷酸酶有远亲关系的酶家族（细菌肌醇多磷酸酶或PTP-IPPs）的分子结构和功能。这些酶从许多不同的肌醇多磷酸盐中去除一个或多个磷酸盐，肌醇多磷酸盐是一类重要的信号转导分子，在许多基本的细胞过程中起作用，包括DNA修复，mRNA输出，植物发育，细胞凋亡和细菌致病性。我们利用各种生物化学和分子生物学技术来研究这些酶的底物特异性、动力学和分子作用机制。特别是，本研究利用x射线晶体学技术来确定ptp - ipp单独和与天然底物复合的原子分辨率结构。这些研究使我们能够可视化酶及其与底物的相互作用。然后利用定点诱变、基于荧光的结合测定和稳态动力学来扩展和完善我们对这些酶的结构和功能的理解。使用这些技术可以解决的具体的、相互关联的问题包括：(1)这些酶如何区分许多自然存在的InsPs？(2)它们的详细动力学机制是什么？它与已被充分研究的蛋白酪氨酸磷酸酶有何不同？(3)能否利用这些酶廉价地生产具有重要生物学意义的inss ？反过来，这些问题的答案将揭示它们在发病机制中的作用以及在真核生物中的作用。

The overall objective of my research program is understanding the molecular structure and function of a recently discovered family of enzymes (bacterial inositol polyphosphatases or PTP-IPPs) that are distantly related to protein tyrosine phosphatases. These enzymes remove one or more phosphates from many different inositol polyphosphates, an important class of signal transduction molecule that has known roles in many essential cellular processes including DNA repair, mRNA export, plant development, apotosis and bacterial pathogenicity. We utilize a variety of biochemical and molecular biology techniques to study the substrate specificity, kinetic and molecular mechanism of action of these enzymes. In particular, this research utilizes X-ray crystallographic techniques to determine atomic resolutions structures of PTP-IPPs alone and in complex with naturally occuring substrates. These studies allow us to visualize the enzyme and its interaction with substrate. Site-directed mutagenesis, fluorescense-based binding assays and steady-state kinetics are then utilized toextend and refine our understanding of the structure and function of these enzymes. Specific, interelated questions of interest that can be addressed using these techniques include: (1) How do these enzymes discriminate between the many naturally occuring InsPs? (2) What is their detailedkinetic mechanism and how does it differ from the well studied protein tyrosine phosphatases? And (3) Can these enzymes be utlized to cheaply produce biologically important InsPs? In turn, the answers to these questions will shed light upon their role in pathogenesis and the role of InsPs in eucaryotes.