title: High-throughput assays of leloir-glycosyltransferase reactions: The applications of rYND1 in glycotechnology.
abstract: Glycosyltransferases (GTs) play a critical role in the enzymatic and chemoenzymatic synthesis of oligosaccharides and glycoconjugates. However, the development of these synthetic approaches has been limited by a lack of sensitive screening methods for the isolation of novel natural GTs or their active variants. Herein, we describe the results of our investigation towards the soluble expression and potential application of the Saccharomyces cerevisiae apyrase YND1. By replacing the hydrophobic transmembrane domain of YND1 with three glycine-serine repeats, this protein was successfully expressed in a soluble form in Escherichia coli. This new protein was then used to develop a two-step nucleoside diphosphate (NDP)-based Leloir-GT high-throughput assay. Purified rYND1 was initially added to a GT reaction to hydrolyze NDP to nucleoside phosphate plus inorganic phosphate, which was determined using a phosphorus molybdenum blue chromogenic reaction. Purified rYND1 was shown to have a positive effect on saccharide synthesis by eliminating the potential by-product inhibition from NDP. Most of the mono-sugar donors used for Leloir-GTs are activated by uridine diphosphate and guanosine diphosphate, which can be catalyzed by rYND1. The rYND1 is amenable to screening methods and could be applied to a wide range of Leloir-GT-catalyzed reactions, therefore representing a remarkable step forward in glycotechnology.
authors.affiliation: Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Li
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Hou
authors.affiliation: Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China; National Glycoengineering Research Center, Shandong University, Jinan 250012, China. Electronic address: fswang@sdu.edu.cn.
authors.forename: Fengshan
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China; National Glycoengineering Research Center, Shandong University, Jinan 250012, China. Electronic address: shengjuzheng@sdu.edu.cn.
authors.forename: Juzheng
authors.initials: J
authors.lastname: Sheng
mesh_terms: D001081:Apyrase / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D001709:Biotechnology / Q000379:methods*; D054794:Glycomics / Q000379:methods*; D016695:Glycosyltransferases / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D016695:Glycosyltransferases; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C023273:uridine diphosphatase; D001081:Apyrase; C506070:Ynd1 protein, S cerevisiae
keywords: Apyrase YND1; Glycosyltransferase reaction; Heterologous soluble expression; High-throughput GT assay; Saccharomyces cerevisiae