title: Characterization of WbiQ: An α1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen.
abstract: Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an α1,2-fucosyltransferase using a radioactivity based assay, thin-layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from 6.5 to 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-β1,3-GalNAc-α-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-α1,2-Gal-β1,3-GalNAc-α-OMe, on a milligram scale.
authors.affiliation: Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Pettit
authors.forename: Thomas
authors.initials: T
authors.lastname: Styslinger
authors.forename: Zhen
authors.initials: Z
authors.lastname: Mei
authors.forename: Weiqing
authors.initials: W
authors.lastname: Han
authors.forename: Guohui
authors.initials: G
authors.lastname: Zhao
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D001789:Blood Group Antigens / Q000096:biosynthesis*; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology*; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D019081:O Antigens / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097631:Galactoside 2-alpha-L-fucosyltransferase
chemical_list: D001789:Blood Group Antigens; D005647:Fucosyltransferases; D019081:O Antigens; D011993:Recombinant Fusion Proteins; D000097631:Galactoside 2-alpha-L-fucosyltransferase
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