Moser Sandra, Hans Gloria, Shukla Anuj, Saiardi Adolfo, Bru Samuel, Taskin Asli Aras, Meisinger Chris, Jessen Henning J
Institute of Organic Chemistry, Albert-Ludwigs-Universität Freiburg Albertstraße 21 79104 Freiburg im Breisgau Germany
Laboratory for Molecular Cell Biology, University College London Gower Street London WC1E 6BT UK.
Chem Sci. 2025 Jun 19. doi: 10.1039/d5sc02166a.
Inorganic polyphosphate (polyP) is a ubiquitous biopolymer composed of multiple orthophosphates connected by energy-rich phosphoanhydride bonds. In organisms, polyP is digested by two types of enzymes: exopolyphosphatases, which shorten the chain from the ends by cleaving off monophosphate units, and endopolyphosphatases, which cut the chain internally. While several continuous methods are available to monitor exopolyphosphatase activity, endopolyphosphatase activity assays are less common and typically involve multiple tedious steps. Here, we introduce FRET-polyP, a novel probe for real-time detection of endopolyphosphatase activity. The FRET assay enabled rapid, highly sensitive, single-step detection of specific endopolyphosphatase activity both from isolated proteins and cell extracts. The simple read-out additionally enabled enzyme inhibitor screening. Furthermore, a novel Mn-dependent endopolyphosphatase activity in baker's yeast was detected in a quadruple mutant, highlighting the ability to screen for metal-dependence of new endopolyphosphatase activity. This approach thus represents a significant addition to existing methodologies, facilitating the discovery and classification of new endopolyphosphatases and their inhibitors to advance our understanding of polyP metabolism and regulation.
无机多聚磷酸盐（多聚P）是一种普遍存在的生物聚合物，由多个通过富含能量的磷酸酐键相连的正磷酸盐组成。在生物体中，多聚P由两种酶消化：外切多聚磷酸酶，通过切割单磷酸单元从链的末端缩短链长；内切多聚磷酸酶，在链内部切割。虽然有几种连续方法可用于监测外切多聚磷酸酶的活性，但内切多聚磷酸酶活性测定较少见，通常涉及多个繁琐步骤。在此，我们介绍FRET-多聚P，一种用于实时检测内切多聚磷酸酶活性的新型探针。FRET测定能够从分离的蛋白质和细胞提取物中快速、高度灵敏、单步检测特定的内切多聚磷酸酶活性。这种简单的读数还能够进行酶抑制剂筛选。此外，在一个四重突变体中检测到面包酵母中一种新的依赖锰的内切多聚磷酸酶活性，突出了筛选新内切多聚磷酸酶活性的金属依赖性的能力。因此，这种方法是对现有方法的重大补充，有助于发现和分类新的内切多聚磷酸酶及其抑制剂，以促进我们对多聚P代谢和调节的理解。
