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J. Biol. Chem., Vol. 280, Issue 44, 36848-36856, November 4, 2005

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A Single Aromatic Amino Acid at the Carboxyl Terminus of Helicobacter pylori 1,3/4 Fucosyltransferase Determines Substrate Specificity^*

Bing Ma, Leon H. Lau, Monica M. Palcic, Bart Hazes1, and Diane E. Taylor, A Medical Scientist with the Alberta Heritage Foundation for Medical Research2

From the 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

Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Gal1,3GlcNAc) or Type II (Gal1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both 1,3 and 1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive 1,3 activity. Our domain swapping study (Ma, B., Wang, G., Palcic, M. M., Hazes, B., and Taylor, D. E. (2003) J. Biol. Chem. 278, 21893–21900) demonstrated that exchange of the hypervariable loops, ³⁴⁷DNPFIFC³⁵³ in 11639FucT and ³⁴⁵CNDAHYSALH³⁵⁴ in UA948FucT, were sufficient to either confer or abolish 1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within ³⁴⁵CNDAHYSALH³⁵⁴ of UA948FucT confer Type I substrate specificity. The Tyr³⁵⁰ Ala mutation dramatically reduced 1,4 activity without lowering 1,3 activity. None of the other alanine substitutions selectively eliminated 1,4 activity. To elucidate how Tyr³⁵⁰ determines 1,4 specificity, mutants Tyr³⁵⁰ Phe, Tyr³⁵⁰ Trp, and Tyr³⁵⁰ Gly were constructed in UA948FucT. These mutations did not decrease 1,3 activity but reduced the 1,4 activity to 61.8%, 78.5%, and 4.7% of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr³⁵⁰, is essential for 1,4 activity. Our data demonstrate that a single amino acid (Tyr³⁵⁰) in the C-terminal hypervariable region of UA948FucT determines Type I acceptor specificity. Notably, a single aromatic residue (Trp) has also been implicated in controlling Type I acceptor preference for human FucT III, but it is located in an N-terminal hypervariable stem domain.

Received for publication, April 22, 2005 , and in revised form, August 12, 2005.

^* This work was supported in part by funding 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.), and an Alberta Ingenuity Scholarship (to L. H. L.). 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.

¹ A Medical Scholar with the Alberta Heritage Foundation for Medical Research.

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.

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This article has been cited by other articles:

				B. Ma, J. L. Simala-Grant, and D. E. Taylor Fucosylation in prokaryotes and eukaryotes Glycobiology, December 1, 2006; 16(12): 158R - 184R. [Abstract] [Full Text] [PDF]

				B. Ma, G. F. Audette, S. Lin, M. M. Palcic, B. Hazes, and D. E. Taylor Purification, Kinetic Characterization, and Mapping of the Minimal Catalytic Domain and the Key Polar Groups of Helicobacter pylori {alpha}-(1,3/1,4)-Fucosyltransferases J. Biol. Chem., March 10, 2006; 281(10): 6385 - 6394. [Abstract] [Full Text] [PDF]