title: Distantly related plant and nematode core α1,3-fucosyltransferases display similar trends in structure-function relationships.
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.
authors.affiliation: Department of Glycobiology, Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská Cesta 9, Bratislava, Slovakia.
authors.forename: Peter
authors.initials: P
authors.lastname: Both
authors.forename: Lukas
authors.initials: L
authors.lastname: Sobczak
authors.forename: Christelle
authors.initials: C
authors.lastname: Breton
authors.forename: Stephan
authors.initials: S
authors.lastname: Hann
authors.forename: Katharina
authors.initials: K
authors.lastname: Nöbauer
authors.forename: Katharina
authors.initials: K
authors.lastname: Paschinger
authors.forename: Stanislav
authors.initials: S
authors.lastname: Kozmon
authors.forename: Ján
authors.initials: J
authors.lastname: Mucha
authors.forename: Iain B H
authors.initials: IB
authors.lastname: Wilson
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology*; D002413:Cations, Divalent; D017124:Conserved Sequence; D057075:Enzyme Assays; D005647:Fucosyltransferases / Q000096:biosynthesis* / Q000737:chemistry; D006031:Glycosylation; D008274:Magnesium; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D040681:Structural Homology, Protein; D053719:Tandem Mass Spectrometry
chemical_list: D002413:Cations, Divalent; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D008274:Magnesium