Glycosyltransferases (GTs) catalyze the formation of regio- and stereospecific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the wfcD gene from the Escherichia coli O141 O-antigen gene cluster encoding an α-1,3-mannosyltransferase that catalyzed the formation of the linkage Man(α1-3)-GlcNAc was biochemically characterized. WfcD 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 glycosidase digestion using the donor substrate GDP-Man and the synthetic acceptor substrate decyl diphosphate 2-acetamido-2-deoxy-α-D-glucopyranose (GlcNAc-PP-De). The kinetic and physiochemical properties and the substrate specificity of WfcD were investigated. WfcD is the first characterized bacterial mannosyltransferase that acts on the Man(α1-3)-GlcNAc linkage. This study enhances our knowledge of the diverse functions of GTs.
Escherichia coli, mass spectrometry, Mannosyltransferase, WfcD
NCBI PubMed ID: 28402841Publication DOI: 10.1016/j.carres.2017.04.003Journal NLM ID: 0043535Publisher: Elsevier
Correspondence: fenglu63@nankai.edu.cn
Institutions: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin, China, The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, 23 Hongda Street, TEDA, Tianjin, China
Methods: SDS-PAGE, ESI-MS/MS, MALDI-MS, biochemical methods, enzyme assay, LC-MS, CID-ESI-IT-MS, kinetics assay
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