Purification, Kinetic Characterization, and Mapping of the Minimal Catalytic Domain and the Key Polar Groups of Helicobacter pylori α-(1,3/1,4)-Fucosyltransferases

doi:10.1074/jbc.M511320200

Purification, Kinetic Characterization, and Mapping of the Minimal Catalytic Domain and the Key Polar Groups of Helicobacter pylori α-(1,3/1,4)-Fucosyltransferases^*

Bing Ma^‡,
Gerald F. Audette^‡,
Shuangjun Lin^§,
Monica M. Palcic^§,
Bart Hazes^‡¹ and
Diane E. Taylor, Medical Scientist of the Alberta Heritage Foundation for Medical Research^‡²

^‡Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2H7 and the ^§Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada

2 To whom correspondence should be addressed: Dept. of Medical Microbiology and Immunology, 1-63 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-4777; Fax: 780-492-7521; E-mail: diane.taylor{at}ualberta.ca.

Abstract

The minimal catalytic domain of α-(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 α-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 α-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 k_cat of 2.3 s^-1 was double that of UA948FucT, which had a k_cat 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 K_m. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori α-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in α1,3 activity, whereas in Type I acceptor this completely abolished α1,4 activity, indicating that this hydroxyl group is a key polar group.

Footnotes

↵3 The abbreviations used are: FucT, fucosyltransferase; GDP-Fuc, guanosine diphosphate β-l-fucose; ΔFucTs, truncated FucTs; Type I, Galβ1,3GlcNAc; Type II, Galβ1,4GlcNAc; Type I-R, Galβ1,3GlcNAc-O(CH₂)₈CO₂CH₃; Type II-R, Galβ1,4GlcNAc-O(CH₂)₈CO₂CH₃; IMAC, immobilized metal affinity chromatography; AIEX, anion exchange chromatography; H-Type II, 2-fucosylated Type II; H-Type I, 2-fucosylated Type I; WT, wild type.
↵4 B. Ma, M. M. Palcic, B. Hazes, and D. E. Taylor, unpublished data.
↵* This work was supported by grants from the Canadian Bacterial Diseases Network Center of Excellence Program (to D. E. T.), the National Sciences and Engineering Research Council, and the Alberta Ingenuity Center for Carbohydrate Science (to M. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains two supplemental tables.
↵1 Scholar of the Alberta Heritage Foundation for Medical Research.
- Received October 18, 2005.
- Revision received December 21, 2005.
The American Society for Biochemistry and Molecular Biology, Inc.