Abstract
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 UDP2-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 UDP2-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 KM, vmax, and kcat 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.
Highlights
The vast molecular diversity and complexity of various oligosaccharide structures and secondary metabolites across all domains of life [1] result from a coherent interplay of enzymatic reactions encompassing the formation and breakdown of glycosidic linkages [2]
The nucleotide-sugar dependent GTs belong to the Leloir enzymes and the glycosyl transfer often occurs at the nucleophilic oxygen of a hydroxyl substituent of the acceptor [2]
The overnight culture was further propagated for 2–3 h in 400 mL of lysogeny broth (LB) supplemented with ampicillin and chloramphenicol until the optical density (OD) at 600 nm reached 0.5–0.6
Summary
The vast molecular diversity and complexity of various oligosaccharide structures and secondary metabolites across all domains of life [1] result from a coherent interplay of enzymatic reactions encompassing the formation and breakdown of glycosidic linkages [2] Enzymes such as glycosyltransferases (GTs) play a crucial role in the enzymatic and chemoenzymatic synthesis of oligosaccharides and glycoconjugates [3]. They catalyze the formation of a glycosidic bond by transferring a sugar moiety from an activated glycosyl donor to a suitable acceptor substrate [2]. This consensus sequence comprises 44 amino acid residues and occurs in all GT1 enzymes [20]
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