A single aromatic amino acid at the carboxyl terminus of Helicobacter pylori {alpha}1,3/4 fucosyltransferase determines substrate specificity.
Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Galbeta1,3GlcNAc) or Type II (Galbeta1,4GlcNAc) 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 alpha1,3 and alpha1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive alpha1,3 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, (347)DNPFIFC(353) in 11639FucT and (345)CNDAHYSALH(354) in UA948FucT, were sufficient to either confer or abolish alpha1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within (345)CNDAHYSALH(354) of UA948FucT confer Type I substrate specificity. The Tyr(350) --> Ala mutation dramatically reduced alpha1,4 activity without lowering alpha1,3 activity. None of the other alanine substitutions selectively eliminated alpha1,4 activity. To elucidate how Tyr(350) determines alpha1,4 specificity, mutants Tyr(350) --> Phe, Tyr(350) --> Trp, and Tyr(350) --> Gly were constructed in UA948FucT. These mutations did not decrease alpha1,3 activity but reduced the alpha1,4 activity to 66.9, 55.6, and 3.1% [corrected] of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr(350), is essential for alpha1,4 activity. Our data demonstrate that a single amino acid (Tyr(350)) 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.
Ma
Bing
B
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Lau
Leon H
LH
Palcic
Monica M
MM
Hazes
Bart
B
Taylor
Diane E
DE
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017931:DNA Primers / Q000737:chemistry; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D014443:Tyrosine; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase