Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif.
The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 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 Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (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) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-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 alpha1,6-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 alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.
Li
Mei
M
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Liu
Xian-Wei
XW
Shao
Jun
J
Shen
Jie
J
Jia
Qiang
Q
Yi
Wen
W
Song
Jing K
JK
Woodward
Robert
R
Chow
Christine S
CS
Wang
Peng George
PG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism; D002850:Chromatography, Gel; D004187:Disaccharides / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D007792:Lactulose / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D015295:Antigens, Tumor-Associated, Carbohydrate; D004187:Disaccharides; D029968:Escherichia coli Proteins; D019081:O Antigens; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; C047464:epilactose; C016467:Thomsen-Friedenreich antigen; D007792:Lactulose; D005647:Fucosyltransferases; D005982:Glutathione Transferase; D007785:Lactose