A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein -Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein -glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein -glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-, , and . To identify the linkage created by the FucT of , we elucidated the full structure of its nine-sugar -glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the  -glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the and  -glycans, the FucT orthologs from , , and each cross-complement the Δ and Δ FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various NP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.