Purification, kinetic characterization, and mapping of the minimal catalytic domain and the key polar groups of Helicobacter pylori alpha-(1,3/1,4)-fucosyltransferases.
The minimal catalytic domain of alpha-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N- and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic alpha-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic alpha-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDP-fucose donor than UA948FucT, and its kcat of 2.3 s(-1) was double that of UA948FucT, which had a kcat value of 1.1 s(-1) for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor Km. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori alpha-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in alpha1,3 activity, whereas in Type I acceptor this completely abolished alpha1,4 activity, indicating that this hydroxyl group is a key polar group.
Ma
Bing
B
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2H7.
Audette
Gerald F
GF
Lin
Shuangjun
S
Palcic
Monica M
MM
Hazes
Bart
B
Taylor
Diane E
DE
D020134:Catalytic Domain / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005647:Fucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D007983:Lewis Blood Group Antigens; D016256:Lewis X Antigen / Q000096:biosynthesis; D009844:Oligosaccharides / Q000096:biosynthesis; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D007983:Lewis Blood Group Antigens; D016256:Lewis X Antigen; C494219:Lewis a oligosaccharide; D009844:Oligosaccharides; D010446:Peptide Fragments; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase