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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686822
• 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蛋白酪氨酸磷酸酶样肌醇磷酸磷酸酶（PTP IPPases）的原核家族的结构，底物特异性和分子作用机制。PTP磷酸肌醇酯酶及其产物肌醇磷酸盐（IP）在农业、生物技术和生命科学领域具有研究价值。越来越多的PTP IPPases是已知的植物病原体毒力因子，其损害或破坏农作物，而具有类似活性的酶（植酸酶）被广泛用作牲畜饲料添加剂，其增加磷酸盐吸收并限制环境损害。此外，PTP IPv6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得令人望而却步的IP的优秀候选者。在真核生物中，IP作为辅因子和第二信使，参与从Ca 2+释放和DNA修复到植物发育和细胞凋亡的许多重要细胞活动。*** 确定单独的PTP IPPases或作为PTP IPPases：底物复合物的类似物的PTP IPPases的X射线晶体学结构，以了解它们的底物特异性和变化的生物学功能。这些结构研究通过荧光结合试验、水解试验、底物特异性试验和定点诱变进行补充，以测试和验证我们的解释。这种方法使我们能够鉴定引起所观察到的PTP IPPases的底物特异性的结构特征，并开发用于大规模生产几种IP的酶和方法。与当地合作者（LB Selinger，生物科学）一起，我们已经积累了PTP IPv6克隆的最大集合之一，并负责这些酶的90%以上的已知原子分辨率结构。这包括来自产生这些酶的原核生物的4个分类学类别中的3个的酶，并且包括对肌醇六磷酸（IP 6或肌醇六磷酸）具有“高活性”的PTP IPPases、具有“高”和“低活性”活性位点的串联重复PTP IPPases和来自噬菌蛭弧菌的毒力因子的实例。*** 已经开发了产生较少磷酸化的IP底物的方法，我们现在正在产生复合物中的较少磷酸化的IP和靶向“窄特异性”PTP IPPases的结构，所述PTP IPPases包括植物病原体毒力因子和含有另外的糖基水解酶结构域的新的酶组。这些研究的目的是了解PTP IPPases的底物特异性和功能。最终，这些信息将有助于开发植物病原体毒力因子的特异性抑制剂，用于注释我们的数据库（http：//PTP-IPPase_phytase. ca）并廉价地产生额外的IP。