title: A fluorescence-based glycosyltransferase assay for high-throughput screening.
abstract: Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.
authors.affiliation: Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea.
authors.forename: Jihye
authors.initials: J
authors.lastname: Ryu
authors.affiliation: Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea.
authors.forename: Min Sik
authors.initials: MS
authors.lastname: Eom
authors.affiliation: Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea.
authors.forename: Wooseok
authors.initials: W
authors.lastname: Ko
authors.affiliation: Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea. Electronic address: mshan@cau.ac.kr.
authors.forename: Min Su
authors.initials: MS
authors.lastname: Han
authors.affiliation: Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea. Electronic address: hslee76@sogang.ac.kr.
authors.forename: Hyun Soo
authors.initials: HS
authors.lastname: Lee
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D007700:Kinetics; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D016695:Glycosyltransferases
keywords: ATP sensor; Glycosyltransferase assay; Glycosyltransferase engineering; High-throughput screening