Structure and Function of PTP-like myo-inositol phosphatases

PTP 样肌醇磷酸酶的结构和功能

• 批准号: RGPIN-2018-06856
• 负责人: Mosimann, Steven
• 金额: $ 2.62万
• 依托单位: University of Lethbridge
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748472
• 关键词: Structure; Function; PTP; like; myo

This research proposal continues to target the structure, substrate specificity and molecular mechanism of action of the prokaryotic family of Protein Tyrosine Phosphatase-like myo-inositol phosphate phosphatases (PTP IPPases). Both the PTP IPPase enzymes and their products, myo-inositol phosphates (IPs), are of research interest in the fields of agriculture, biotechnology and the life sciences. A growing number of the PTP IPPases are known phytopathogen virulence factors that damage or destroy agricultural crops while enzymes with similar activities (phytases) are widely used as livestock feed additives that increase phosphate absorption and limit environmental damage. Further, divergent members of the PTP IPPase family produce a wide variety of IPs, making them excellent candidates for the production of commercially unavailable or prohibitively expensive IPs. In eukaryotes, IPs serve as cofactors and second messengers that participate in numerous important cellular activities from Ca2+ release and DNA repair to plant development and apoptosis. X-ray crystallographic structures of PTP IPPases alone or as analogs of the PTP IPPase:substrate complex are determined in order to understand their substrate specificity and varied, biological functions. These structural studies are supplemented by fluorescent binding assays, hydrolysis assays, substrate specificity assays and site-directed mutagenesis to test and validate our interpretations. This approach has allowed us identify structural features that give rise to the observed substrate specificity of PTP IPPases and develop both enzymes and methods for the large scale production of several IPs. Together with a local collaborator (LB Selinger, Biological Sciences) we have amassed one of the largest collections of PTP IPPase clones and are responsible for more than 90% of the known atomic resolution structures of these enzymes. This includes enzymes from 3 of the 4 taxonomic classes of prokaryotes that produce these enzymes and includes examples of PTP IPPases with 'high activity' towards myo-inositol hexakisphosphate (IP6 or phytate), a tandemly repeated PTP IPPase with both 'high' and 'low activity' active sites and a virulence factor from Bdellovibrio bacteriovorans. Having developed methods to generate less-phosphorylated IP substrates, we are now generating structures in complex the less-phosphorylated IPs and targeting 'narrow specificity' PTP IPPases that include the phytopathogen virulence factors and novel group of enzymes that contain an additional glycosyl hydrolase domain. The goal of these studies is to understand the substrate specificity and function of PTP IPPases. Ultimately, this information will contribute to the development of specific inhibitors of phytopathogen virulence factors, be used to annotate our database (http://PTP-IPPase_phytase.ca) and cheaply generate additional IPs.

这项研究计划继续针对原核蛋白酪氨酸磷酸酶样肌醇磷酸酶家族(PTP IPPase)的结构、底物特异性和分子作用机制。PTP IPPase酶及其产物肌醇磷酸(IP)在农业、生物技术和生命科学领域具有重要的研究价值。越来越多的PTP IPPase是已知的危害或破坏农作物的植物病原体毒力因子，而具有类似活性的酶(植酸酶)被广泛用作家畜饲料添加剂，增加磷酸盐的吸收，限制环境破坏。此外，PTP IPPase家族的不同成员可以产生各种各样的IP，使它们成为生产商业上无法获得的或昂贵得令人望而却步的IP的极佳候选者。在真核生物中，IP作为辅因子和第二信使参与了许多重要的细胞活动，从钙释放和DNA修复到植物发育和细胞凋亡。测定了PTP IPPase单独或作为PTP IPPase：底物复合体的类似物的X射线晶体结构，以了解它们的底物特异性和不同的生物学功能。这些结构研究辅之以荧光结合分析、水解分析、底物特异性分析和定点突变，以测试和验证我们的解释。这种方法使我们能够确定导致观察到的PTP IPPase底物特异性的结构特征，并开发出用于大规模生产几个IP的酶和方法。与当地的一位合作者(生物科学的LB Selinger)一起，我们已经积累了最大的PTP IPPase克隆之一，并负责这些酶90%以上的已知原子分辨结构。这包括产生这些酶的4个分类类别中的3个原核生物的酶，包括对肌醇六磷酸(IP6或植酸盐)具有高活性的PTP IPPase的例子，具有“高”和“低活性”活性位点的简单重复的PTP IPPase，以及来自Bdellovibrio细菌体的毒力因子。在开发出生成磷酸化程度较低的IP底物的方法后，我们现在正在生成复杂的磷酸化程度较低的IP的结构，并针对包括植物病原体毒力因子和包含额外糖基水解酶结构域的新型酶的“狭义特异性”PTP IPPase。这些研究的目的是了解PTP IPPase的底物特异性和功能。最终，这些信息将有助于开发特定的植物病原体毒力因子抑制剂，用于注释我们的数据库(http://PTP-IPPase_phytase.ca)和廉价地产生额外的IP。