A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes
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Author(s)
Das, Debasis
Walvoort, Maria Theresia Cornelia
Lukose, Vinita
Imperiali, Barbara
Date Issued
September 2016
Journal
Scientific Reports
Publisher
Nature Publishing Group
Citation
Das, Debasis, Marthe. T. C. Walvoort, Vinita Lukose, and Barbara Imperiali. “A Rapid and Efficient Luminescence-Based Method for Assaying Phosphoglycosyltransferase Enzymes.” Scientific Reports 6, no. 1 (September 14, 2016).
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Final published version
Abstract
Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD+ generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1038/srep33412
