title: High-throughput colorimetric assays for nucleotide sugar formation and glycosyl transfer.
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.
authors.affiliation: Pharmaceutical Sciences Division, School of Pharmacy, Wisconsin Center for Natural Products Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Gantt
authors.forename: Jon S
authors.initials: JS
authors.lastname: Thorson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D055162:Biocatalysis; D003124:Colorimetry / Q000379:methods*; D055808:Drug Discovery; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D009596:Nitrophenols / Q000032:analysis; D009711:Nucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013303:Streptomyces antibioticus / Q000737:chemistry / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D006027:Glycosides; D009596:Nitrophenols; D009711:Nucleotides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C519503:OleD protein, Streptomyces antibioticus; C548837:2-chloro-4-nitrophenol