Characterization of a novel (α1,2-fucosyltransferase of Escherichia coli O128:B12 and functional investigation of its common motif

The wbsJ gene from Escherichia coli O128:B12 encodes an (alpha 1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an (alpha 1,2 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 Gal beta 1,3GalNAc (T antigen), Gal beta 1,4Man and Gal beta 1,4Glc (lactose) being better acceptors than Gal beta-O-Me and galactose. Gal beta 1,41Fru (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 K values of 7.81 mM and 13.26 mM, respectively. However, the k(cat)/(app)&K-m value of lactose (6.36 M-1 center dot min(-1)) is two times lower than that of lactulose (13.39 M-1 center dot min(-1)). In addition, the alpha-1,2-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 K-i 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 H(152)xR(154)R(155)xD(157). in contrast to (alpha 1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R-154 and D-157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha 1,2-fucosyltransferases and alpha 1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Gal beta-O-Me and Gal beta 1,4Glc beta-N-3 as acceptors.