Juliano Alves 1 , Jacquelyn Hennek 1,two , Mentioned A. Goueli 1,three and Hicham Zegzouti 1, 2Promega Corporation, R D Department, 2800 Woods Hollow Road, Madison, WI 53719, USA; laurie.engel@promega (L.E.); juliano.alves@promega (J.A.); jhennek@exactsciences (J.H.); said.goueli@promega (S.A.G.) Exact Sciences Corporation, 5505 Endeavor Lane, Madison, WI 53719, USA Department of Pathology and Laboratory Medicine, University of Wisconsin School of D4 Receptor Agonist custom synthesis Medicine and Public Overall health, Madison, WI 53719, USA Correspondence: hicham.zegzouti@promegaCitation: Engel, L.; Alves, J.; Hennek, J.; Goueli, S.A.; Zegzouti, H. Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors. Molecules 2021, 26, 6230. doi.org/10.3390/ moleculesAbstract: Classic glycosyltransferase (GT) activity assays are usually not simply configured for rapid detection nor for higher throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. Inside a typical glycosyltransferase biochemical reaction, two merchandise are generated, a glycosylated item along with a nucleotide released from the sugar donor substrate. As a result, an assay that detects the nucleotide might be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays depending on UDP, GDP, CMP, and UMP detection. Each of these assays are performed within a one-step detection that relies on converting the nucleotide solution to ATP, then to bioluminescence utilizing firefly luciferase. These assays are very sensitive, robust and resistant to chemical interference. Many applications of these assays are presented, like studies around the specificity of sugar transfer by diverse GTs plus the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Moreover, their utility in screening for EP Agonist custom synthesis particular 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 big number of GTs and might have a important effect on diverse regions of Glycobiology investigation. Key phrases: nucleotide assays; bioluminescence; sugar substrate; fucosyltransferase; OGT; inhibitorAcademic Editor: Stefan Janecek Received: 16 September 2021 Accepted: 12 October 2021 Published: 15 October1. Introduction Glycosyltransferases (GT) represent a big loved ones of enzymes that belong to a welldefined enzymatic network that orchestrates the formation and upkeep of complex carbohydrate structures located abundantly in all living organisms [1]. Employing activated sugars as donor substrates, glycosyltransferases transfer the sugar moiety to an array of acceptor substrates of many chemical natures, including proteins, lipids, sugars, nucleic acids, and modest molecules [2]. By far the most popular donor substrates utilised by glycosyltransferases are nucleotide-activated sugars, like UDP-, GDP-, and CMP-sugars, but they may also use lipid sugar phosphates (e.g., dolichol phosphate sugar), and unsubstituted phosphates. Glycosyltransferases that use nucleotide-activated sugars are known as Leloir enzymes, in honor on the 1970 chemistry Nobel Prize winner Luis F. Leloir, who discovered the first sugar nucleotide [3]. Because of the value of the various oligosaccharide structures to cell f
