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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714505
• 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.

本研究计划继续针对蛋白酪氨酸磷酸酶样肌醇磷酸酶（PTP IPPases）原核家族的结构、底物特异性和分子作用机制进行研究。PTP IPPase及其产物肌醇磷酸（myo-inositol phosphate, IPs）在农业、生物技术和生命科学等领域具有重要的研究价值。越来越多的PTP ippase被认为是破坏或摧毁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）被广泛用作牲畜饲料添加剂，以增加磷酸盐吸收和限制环境破坏。此外，PTP IPPase家族的不同成员生产各种各样的ip，使它们成为生产商业上不可用或昂贵的ip的绝佳候选。在真核生物中，IPs作为辅助因子和第二信使参与了从Ca2+释放和DNA修复到植物发育和凋亡的许多重要细胞活动。