High-throughput colorimetric assays for nucleotide sugar formation and glycosyl transfer

Richard W. Gantt, Jon S. Thorson

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

8 Scopus citations

Abstract

Glycosyltransferases are ubiquitous in nature, catalyzing glycosidic bond formation in the context of an enormous range of substrates, which include all major classes of biological molecules. Because this wide range of substrates lacks a shared, distinguishable feature that can be altered by glycosyl transfer, general assays for detection of glycosyltransferase activity have long been largely limited to low-throughput methods. Of those high-throughput assays reported in the literature, many are confined to specific glycosyl transfer reactions with modified aglycon acceptors selected for their unique analytical properties. Herein are described a series of protocols centered on the use of 2-chloro-4-nitrophenyl glycoside donors and the reversibility of glycosyltransferase-catalyzed reactions to enable a colorimetric assay for the formation of sugar nucleotides, coupled reaction systems for the glycodiversification of small molecules, and a general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering, and other fundamental sugar nucleotide-dependent investigations.

Original languageEnglish
Title of host publicationMethods in Enzymology
Pages345-360
Number of pages16
DOIs
StatePublished - 2012

Publication series

NameMethods in Enzymology
Volume516
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Colorimetric assay
  • Glycosyl transfer
  • Glycosyltransferase
  • Nucleotide diphosphate sugar

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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