The wbsJ gene from Escherichia coli O128:B12 encodes an α1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an α1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galβ1,3GalNAc (T antigen), Galβ1,4Man and Galβ1,4Glc (lactose) being better acceptors than Galβ-O-Me and galactose. Galβ1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M-1.min-1) is two times lower than that of lactulose (13.39 M-1.min-1). In addition, the α1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to α1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both α1,2-fucosyltransferases and α1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galβ-O-Me and Galβ1,4Glcβ-N3 as acceptors
O-antigen, Escherichia coli, enzymatic synthesis, a-1, 2-fucosyltransferase, wbsJ gene
NCBI PubMed ID: 18078329Journal NLM ID: 0370623Publisher: American Chemical Society
Correspondence: wang.892@osu.edu
Institutions: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, and Department of Biochemistry and Chemistry, The Ohio State University, Columbus, Ohio 43210
Methods: 13C NMR, 1H NMR, ESI-MS, NMR-1D, genetic methods, biochemical methods
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