A screening approach unveils an unknown Mn2+-dependent endopolyphosphatase activity in yeast

Abstract

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-polyP8, 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 Mn2+-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.

Graphical abstract: A screening approach unveils an unknown Mn2+-dependent endopolyphosphatase activity in yeast

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Article information

Article type
Edge Article
Submitted
20 Mar 2025
Accepted
18 Jun 2025
First published
19 Jun 2025
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2025, Advance Article

A screening approach unveils an unknown Mn2+-dependent endopolyphosphatase activity in yeast

S. Moser, G. Hans, A. Shukla, A. Saiardi, S. Bru, A. A. Taskin, C. Meisinger and H. J. Jessen, Chem. Sci., 2025, Advance Article , DOI: 10.1039/D5SC02166A

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