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Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases

The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of h...

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Autores principales: McGraphery, Kate, Schwab, Wilfried
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139940/
https://www.ncbi.nlm.nih.gov/pubmed/32210023
http://dx.doi.org/10.3390/ijms21062208
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author McGraphery, Kate
Schwab, Wilfried
author_facet McGraphery, Kate
Schwab, Wilfried
author_sort McGraphery, Kate
collection PubMed
description The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of high-throughput (HTP) available methods, hinders the extrapolation of further novel applications. In this study, the applicability of new GT-assays suitable for HTP screening was tested and compared with regard to harmlessness, robustness, cost-effectiveness and reproducibility. The UDP-Glo GT-assay, Phosphate GT Activity assay, pH-sensitive GT-assay, and UDP(2)-TR-FRET assay were applied and tailored to plant UDP GTs (UGTs). Vitis vinifera (UGT72B27) GT was subjected to glycosylation reaction with various phenolics. Substrate screening and kinetic parameters were evaluated. The pH-sensitive assay and the UDP(2)-TR-FRET assay were incomparable and unsuitable for HTP plant GT-1 family UGT screening. Furthermore, the UDP-Glo GT-assay and the Phosphate GT Activity assay yielded closely similar and reproducible K(M), v(max), and k(cat) values. Therefore, with the easy experimental set-up and rapid readout, the two assays are suitable for HTP screening and quantitative kinetic analysis of plant UGTs. This research sheds light on new and emerging HTP assays, which will allow for analysis of novel family-1 plant GTs and will uncover further applications.
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spelling pubmed-71399402020-04-13 Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases McGraphery, Kate Schwab, Wilfried Int J Mol Sci Article The ability of glycosyltransferases (GTs) to reduce volatility, increase solubility, and thus alter the bioavailability of small molecules through glycosylation has attracted immense attention in pharmaceutical, nutraceutical, and cosmeceutical industries. The lack of GTs known and the scarcity of high-throughput (HTP) available methods, hinders the extrapolation of further novel applications. In this study, the applicability of new GT-assays suitable for HTP screening was tested and compared with regard to harmlessness, robustness, cost-effectiveness and reproducibility. The UDP-Glo GT-assay, Phosphate GT Activity assay, pH-sensitive GT-assay, and UDP(2)-TR-FRET assay were applied and tailored to plant UDP GTs (UGTs). Vitis vinifera (UGT72B27) GT was subjected to glycosylation reaction with various phenolics. Substrate screening and kinetic parameters were evaluated. The pH-sensitive assay and the UDP(2)-TR-FRET assay were incomparable and unsuitable for HTP plant GT-1 family UGT screening. Furthermore, the UDP-Glo GT-assay and the Phosphate GT Activity assay yielded closely similar and reproducible K(M), v(max), and k(cat) values. Therefore, with the easy experimental set-up and rapid readout, the two assays are suitable for HTP screening and quantitative kinetic analysis of plant UGTs. This research sheds light on new and emerging HTP assays, which will allow for analysis of novel family-1 plant GTs and will uncover further applications. MDPI 2020-03-23 /pmc/articles/PMC7139940/ /pubmed/32210023 http://dx.doi.org/10.3390/ijms21062208 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
McGraphery, Kate
Schwab, Wilfried
Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
title Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
title_full Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
title_fullStr Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
title_full_unstemmed Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
title_short Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases
title_sort comparative analysis of high-throughput assays of family-1 plant glycosyltransferases
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139940/
https://www.ncbi.nlm.nih.gov/pubmed/32210023
http://dx.doi.org/10.3390/ijms21062208
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