Abstract
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.
Highlights
Both ␣-(1,2)- and ␣-(1,3/1,4)-FucTs have been identified and characterized in Helicobacter pylori (21–23, 25–28)
For H. pylori FucT from strain DMS6709, primarily an ␣-(1,4)-FucT, 3Ј-sialyl-Type I is an excellent acceptor (25). This suggests that the hydroxyl at C-3 of galactose is not required for the recognition by H. pylori ␣-(1,3/1,4)-FucTs
Our current data demonstrate that the C terminus, but not the N terminus, of H. pylori ␣-(1,3/1,4)-FucTs can be truncated without significant loss in activity
Summary
Materials—Primers for constructing the truncated FucTs (⌬FucTs) were synthesized by Invitrogen. Radiochemical Assays of ⌬FucTs—Enzyme activities of each truncated FucT in crude cell extracts was assayed with donor GDP-Fuc at 200 M, GDP-3[H]Fuc at 0.2 M (ϳ60,000 dpm), and Type II-R or Type I-R acceptors at 1.8 or 7.5 mM, respectively (21, 23, 24). Single Substrate Kinetics of WT and 116391–441 and UA9481–428 in Cell Crude Extracts—The acceptor and donor kinetics for the fulllength and 11639FucT1–441 and UA948FucT1–428 in cell crude extracts were carried out as described previously (24, 44). Synthesis of Lewis X or Lewis A Using Purified 116391–441 and UA9481–428—Type II-R (2 mg, 3.6 mol) and GDP-Fuc (3.5 mg, 5.4 mol) were used for Lewis X synthesis with purified 11639FucT1–441 in 1.5 ml of HEPES buffer (20 mM, pH 7.0) containing 100 mM NaCl, 35 mM MgCl2, 5 mg mlϪ1 bovine serum albumin, 20 mM MnCl2. The synthesized products, Lewis X and Lewis A, were characterized by 1H NMR spectroscopic analysis
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