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Type of publication: Journal Article
Type of document: Full Paper

Year: 2011

Authors: Both, P; Sobczak, L; Breton, C; Hann, S; Nöbauer, K; Paschinger, K; Kozmon, S; Mucha, J; Wilson, IB

Title: Distantly related plant and nematode core α1,3-fucosyltransferases display similar trends in structure-function relationships.

Source: Glycobiology. 2011; 21(11):1401-1415



Authors Vetmeduni Vienna:

Nöbauer Katharina

Vetmed Research Units
VetCore


Abstract:
Here, we present a comparative structure-function study of a nematode and a plant core α1,3-fucosyltransferase based on deletion and point mutations of the coding regions of Caenorhabditis elegans FUT-1 and Arabidopsis thaliana FucTA (FUT11). In particular, our results reveal a novel "first cluster motif" shared by both core and Lewis-type α1,3-fucosyltransferases of the GT10 family. To evaluate the role of the conserved serine within this motif, this residue was replaced with alanine in FucTA (S218) and FUT-1 (S243). The S218A replacement completely abolished the enzyme activity of FucTA, while the S243A mutant of FUT-1 retained 20% of the "wild-type" activity. Based on the results of homology modeling of FucTA, other residues potentially involved in the donor substrate binding were examined, and mutations of N219 and R226 dramatically affected enzymatic activity. Finally, as both FucTA and FUT-1 were shown to be N-glycosylated, we examined the putative N-glycosylation sites. While alanine replacements at single potential N-glycosylation sites of FucTA resulted in a loss of up to 80% of the activity, a triple glycosylation site mutant still retained 5%, as compared to the control. In summary, our data indicate similar trends in structure-function relationships of distantly related enzymes which perform similar biochemical reactions and form the basis for future work aimed at understanding the structure of α1,3-fucosyltransferases in general.

Keywords Pubmed: Amino Acid Motifs
Amino Acid Sequence
Amino Acid Substitution
Animals
Arabidopsis/enzymology*
Binding Sites
Caenorhabditis elegans/enzymology*
Cations, Divalent
Conserved Sequence
Enzyme Assays
Fucosyltransferases/biosynthesis*
Fucosyltransferases/chemistry
Glycosylation
Magnesium
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptide Fragments/chemistry
Protein Structure, Tertiary
Recombinant Proteins/biosynthesis
Recombinant Proteins/chemistry
Structural Homology, Protein
Tandem Mass Spectrometry


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