Biochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3

•WclR is the first bacterial GalT characterized that acts on the linkage Gal α 1, 3-GlcNAc.•This study enhanced our knowledge of the diverse functions of GTs and provided a possible novel enzyme source for pharmaceutical applications.•This is a novel identification method for glycosyltransferase by CID-ESI-IT-MSn and galactosidase digestion. [Display omitted] Glycosyltransferases (GTs) catalyze the formation of regio- and stereo-specific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the gene wclR from the Escherichia coli O3 O-antigen gene cluster that encodes an α 1, 3-galactosyltransferase (GalT) that acts on the linkage Gal α 1, 3-GlcNAc was biochemically characterized. WclR was expressed in E. coli BL21 (DE3), and the enzymatic product was identified by liquid chromatography-mass spectrometry (LC-MS), collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn) and galactosidase digestion, using UDP-Gal as the donor substrate and the synthetic acceptor substrate GlcNAc-PP-De (decyl diphosphate N-acetylglucosamine). The physiochemical properties and the substrate specificity of WclR were investigated. WclR is the first bacterial GalT characterized that acts on the linkage Gal α 1, 3-GlcNAc. This study enhanced our knowledge of the diversified functions of GTs and provided a novel enzyme source for possible pharmaceutical application.