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一系列酶(细菌肌醇多磷酸酶或PTP-IPP)的分子结构和功能，这些酶与蛋白质酪氨酸磷酸酶有较远的亲缘关系。这些酶从许多不同的肌醇多磷酸中去除一种或多种磷酸盐，肌醇多磷酸是一类重要的信号转导分子，已知在许多重要的细胞过程中发挥作用，包括DNA修复、mRNA输出、植物发育、细胞凋亡和细菌致病。我们利用各种生化和分子生物学技术来研究这些酶的底物专一性、动力学和分子机制。特别是，这项研究利用X射线结晶学技术来确定PTP-IPPS单独的以及与自然生成的底物形成的络合物的原子分辨率结构。这些研究使我们能够直观地了解酶及其与底物的相互作用。然后利用定点突变、基于荧光的结合分析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特定的、相关的问题包括：(1)这些酶如何区分许多自然产生的INSP？(2)它们详细的动力学机制是什么，它与研究得很好的蛋白质酪氨酸磷酸酶有什么不同？以及(3)这些酶能否被用来廉价地生产具有重要生物学意义的Insps？反过来，这些问题的答案将有助于阐明它们在发病机制中的作用以及INSPs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.