Exploring the sequence-function space of microbial fucosidases

Microbial α-l-fucosidases catalyse the hydrolysis of terminal α-l-fucosidic linkages with diverse substrate/linkage specificities and can be used in transglycosylation reactions to synthesise oligosaccharides. Based on sequence identity, α-l-fucosidases have been classified in distinct glycoside hydrolases (GHs) families in the carbohydrate-active enzymes (CAZy) database. Here, we explored the sequence-function space of fucosidases from GH29 family. Based on sequence similarity network (SSN) analyses, 16 GH29 α-l-fucosidases were selected for functional characterisation. Using activity assays combined with HPAEC-PAD and LC-FD-MS/MS analyses, we determined the substrate and linkage specificities of these enzymes against a range of defined oligosaccharides and glycoconjugates, revealing a range of specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated ligands. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1-6 linkages and FA2G2 N-glycan was further determined by X-ray crystallography and saturation transfer difference NMR. TLC combined with electrospray ionization – MS and NMR confirmed the capacity of this enzyme to carry out transfucosylation reactions with GlcNAc and Fuc1,3GlcNAc as acceptors. Taken together, these experimental data validate the use of SSN as a reliable bioinformatics approach to predict the substrate specificity and transfucosylation activity of GH29 fucosidases.