Glycobiology Advance Access published online on September 29, 2004
Glycobiology, doi:10.1093/glycob/cwh157
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1 Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA
* To whom correspondence should be addressed. E-mail: macher{at}sfsu.edu.
Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif by Martin et al. (Martin, et al., 1997) which 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, in those cases in which the mutant protein had sufficient activity, kinetically characterized. Two of the mutant proteins were inactive, six had less than one percent of wild type activity, and four had approximately ten to fifty percent of wild type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (five to fifteen times) for GDP-fucose than FucT VI wild type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least ten times larger than wild type FucT VI for the acceptor substrate, with only a slightly larger (two to three 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-fucosyltransferase 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.
Revised August 31, 2004
Accepted September 21, 2004
ORIGINAL ARTICLES
Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates
2 Vertex Pharmaceuticals (Europe) Ltd., Abingdon, Oxfordshire OX14 4RY, UK
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