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

McGraphery, Kate; Schwab, Wilfried

doi:10.3390/ijms21062208

Cited by 14 publications

(16 citation statements)

References 54 publications

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“…Determining the function of a GT currently requires correct identification of both donor and acceptor substrates in addition to the development of an assay for product identification. Some commercial (UDP/CMP/GDP-Glo™, Promega) and reported glycosylation assays are based on detection of UDP or GDP released during the reaction through coupling to another reaction [ 5 , 6 ] making them more suitable for high throughput studies, but they still depend on acceptor substrates. Often acceptor substrates are not only unknown, but candidate substrates are generally not available from commercial and other sources.…”

Section: Introductionmentioning

confidence: 99%

Microscale thermophoresis as a powerful tool for screening glycosyltransferases involved in cell wall biosynthesis

et al. 2020

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Background: Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of glycosyltransferases are very low throughput and acceptor substrates are generally not available. Results: We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein-ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. Conclusions: This method is rapid and sensitive to allow determination of both donor and acceptor substrates of glycosyltransferases. MST enables high throughput screening of glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of glycosyltransferase function.

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Section: Introductionmentioning

confidence: 99%

Microscale thermophoresis as a powerful tool for screening glycosyltransferases involved in cell wall biosynthesis

et al. 2020

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“…While we used known glycosyltransferases to demonstrate donor/acceptor substrate preferences, others have shown the importance of these assays in unlocking the glycosylation specificity of GTs of unknown mechanisms [ 42 , 43 , 44 , 45 ], characterizing the biochemical features of difficult-to-assay PGTs and their homologs from different species [ 46 ], or screen various naturally-occurring substrates of plant UGTs [ 47 ]. Using UDP-Glo assay, TMEM5, which is a membrane protein required for the functional glycosylation of dystroglycan, was shown to be a xylosyltransferase by testing multiple UDP-sugars, and only the UDP-Xylose was used by the protein [ 44 ].…”

Section: Resultsmentioning

confidence: 99%

Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors

et al. 2021

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Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.

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“…In order to characterize the enzyme activity of UGT93Y1 in more detail with respect to its particular substrate specificity towards menthol, a quantitative substrate screening was performed using the UDP Glo™ Glycosyltransferase assay [ 40 ]. In this assay, the enzymatically released amount of UDP is determined to calculate relative activities.…”

Section: Resultsmentioning

confidence: 99%

“…The reaction was stopped by adding 12.5 µL 0.6 M HCl and further neutralization with 1 M TRIZMA base. Five µL of the GT reaction was pipetted to a 384 well plate [ 40 ]. The calculation of kinetic data was performed with KaleidaGraph ( ; v4.5.4; accessed on 8 September 2021).…”

Section: Methodsmentioning

confidence: 99%

Glucosylation of (±)-Menthol by Uridine-Diphosphate-Sugar Dependent Glucosyltransferases from Plants

et al. 2021

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Menthol is a cyclic monoterpene alcohol of the essential oils of plants of the genus Mentha, which is in demand by various industries due to its diverse sensorial and physiological properties. However, its poor water solubility and its toxic effect limit possible applications. Glycosylation offers a solution as the binding of a sugar residue to small molecules increases their water solubility and stability, renders aroma components odorless and modifies bioactivity. In order to identify plant enzymes that catalyze this reaction, a glycosyltransferase library containing 57 uridine diphosphate sugar-dependent enzymes (UGTs) was screened with (±)-menthol. The identity of the products was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Five enzymes were able to form (±)-menthyl-β-d-glucopyranoside in whole-cell biotransformations: UGT93Y1, UGT93Y2, UGT85K11, UGT72B27 and UGT73B24. In vitro enzyme activity assays revealed highest catalytic activity for UGT93Y1 (7.6 nkat/mg) from Camellia sinensis towards menthol and its isomeric forms. Although UGT93Y2 shares 70% sequence identity with UGT93Y1, it was less efficient. Of the five enzymes, UGT93Y1 stood out because of its high in vivo and in vitro biotransformation rate. The identification of novel menthol glycosyltransferases from the tea plant opens new perspectives for the biotechnological production of menthyl glucoside.

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

Cited by 14 publications

References 54 publications

Microscale thermophoresis as a powerful tool for screening glycosyltransferases involved in cell wall biosynthesis

Microscale thermophoresis as a powerful tool for screening glycosyltransferases involved in cell wall biosynthesis

Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors

Glucosylation of (±)-Menthol by Uridine-Diphosphate-Sugar Dependent Glucosyltransferases from Plants

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