A Novel Fluorescent Probe for Retaining Galactosyltransferases

Abstract

Glycosyltransferases (GTs) are a large class of carbohydrate-active enzymes that are involved, in both pro- and eukaryotic organisms, in numerous important biological processes, from cellular adhesion to carcinogenesis. GTs have enormous potential as molecular targets for chemical biology and drug discovery. For the full realisation of this potential, operationally simple and generally applicable GT bioassays, especially for inhibitor screening, are indispensable tools. In order to facilitate the development of GT high-throughput screening assays for the identification of GT inhibitors, we have developed novel, fluorescent derivatives of UDP-galactose (UDP-Gal) that are recognised as donor analogues by several different retaining galactosyltransferases (GalTs). We demonstrate for one of these derivatives that fluorescence emission is quenched upon specific binding to individual GalTs, and that this effect can be used as the read-out in ligand-displacement experiments. The novel fluorophore acts as an excellent sensor for several different enzymes and is suitable for the development of a new type of GalT bioassay, whose modular nature and operational simplicity will significantly facilitate inhibitor screening. Importantly, the structural differences between the natural donor UDP-Gal and the new fluorescent derivatives are minimal, and the general assay principle described herein may therefore also be applicable to other GalTs and/or proteins that use nucleotides or nucleotide conjugates as their cofactor.

Using simple donors to drive the equilibria of glycosyltransferase-catalyzed reactions

“…5). The ability to do so presents one of the first truly general continuous GT assays as the colorimetric read-out directly correlates to NDP-sugar usage in such reactions and thereby avoids the need for additional manipulations or specialized probes commonly associated with conventional assays for glycosidic bond formation (39–41) . To demonstrate this approach, a set of 50 ( 62–111 ) medicinally relevant compounds were screened with the single GT-catalyzed reaction in a high throughput format.…”

Recent advances in the enzymatic synthesis of sugar-nucleotides using nucleotidylyltransferases and glycosyltransferases

“…8 This provided enzymatic access to a new class of 5-position modified sugar-nucleotides, which had previously demonstrated promise as GT inhibitors 9 and as fluorescent tools for GT assay development. 10 Their strategy employed a small panel of 5position modified UTPs and Glc 1-phosphate, incubated with UDP-glucose pyrophosphorylase (GalU) to generate, in situ, a modified UDP-Glc in catalytic quantity, to be continuously recycled by GalU. Subsequently, Gal 1-phosphate uridylyltransferase (GalPUT) catalysed the reaction of base-modified UDP-Glc into Glc 1-phosphate and the corresponding UDP-Gal.…”

Nucleoside Diphosphate Sugar Analogues that Target Glycosyltransferases

“…Compounds 67 are fluorescent derivatives that are recognizedb ys everalr etaining galactosyltransferases (GalTs), which act as excellent sensors. [60] Enzymological studies with representative bovine a-(1,3)-GalT showed that, whereas the turnovers of compounds 67 a-67 d were considerably lower than for UDPÀGal( K cat = 0.98 s À1 ), the Michaelis-Menten constants of the analogues were of as imilar order of magnitude as for UDPÀGal (K m = 118(AE 14) mm).…”