A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes
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 o...
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2017
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| Online Access: | http://hdl.handle.net/1721.1/109393 https://orcid.org/0000-0001-8967-3348 https://orcid.org/0000-0003-3761-3743 https://orcid.org/0000-0002-5749-7869 |
| _version_ | 1826191419745763328 |
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| author | Das, Debasis Walvoort, Maria Theresia Cornelia Lukose, Vinita Imperiali, Barbara |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Das, Debasis Walvoort, Maria Theresia Cornelia Lukose, Vinita Imperiali, Barbara |
| author_sort | Das, Debasis |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T08:55:35Z |
| format | Article |
| id | mit-1721.1/109393 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:55:35Z |
| publishDate | 2017 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1093932022-09-26T09:16:16Z A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes Das, Debasis Walvoort, Maria Theresia Cornelia Lukose, Vinita Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Das, Debasis Walvoort, Maria Theresia Cornelia Lukose, Vinita Imperiali, Barbara 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. National Institutes of Health (U.S.) (GM-039334) 2017-05-26T19:48:38Z 2017-05-26T19:48:38Z 2016-09 2016-07 Article http://purl.org/eprint/type/JournalArticle 2045-2322 http://hdl.handle.net/1721.1/109393 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). https://orcid.org/0000-0001-8967-3348 https://orcid.org/0000-0003-3761-3743 https://orcid.org/0000-0002-5749-7869 en_US http://dx.doi.org/10.1038/srep33412 Scientific Reports Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/ application/pdf Nature Publishing Group Nature |
| spellingShingle | Das, Debasis Walvoort, Maria Theresia Cornelia Lukose, Vinita Imperiali, Barbara A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes |
| title | A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes |
| title_full | A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes |
| title_fullStr | A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes |
| title_full_unstemmed | A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes |
| title_short | A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes |
| title_sort | rapid and efficient luminescence based method for assaying phosphoglycosyltransferase enzymes |
| url | http://hdl.handle.net/1721.1/109393 https://orcid.org/0000-0001-8967-3348 https://orcid.org/0000-0003-3761-3743 https://orcid.org/0000-0002-5749-7869 |
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