Glycobiology Advance Access originally published online on September 29, 2004
Glycobiology 2005 15(2):165-175; doi:10.1093/glycob/cwh157
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Glycobiology vol. 15 no. 2 © Oxford University Press 2005; all rights reserved.
Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates
3 Department of Chemistry and Biochemistry, 1600 Holloway Ave., San Francisco State University, San Francisco, CA 94132, and 4 Vertex Pharmaceuticals (Europe) Ltd., Abingdon, Oxfordshire OX14 4RY, United Kingdom
2 To whom correspondence should be addressed; e-mail: macher{at}sfsu.edu
Received on May 26, 2004; revised on August 31, 2004; accepted on September 21, 2004
Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had 
10–50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2–3 times) Km for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix–loop–helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.
Key words: alanine scanning mutagenesis / alpha 1,3-fucosyltransferase motif / function / molecular modeling / structure
1 Present address: Department of Bio-organic Chemistry I, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
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