Structural basis for acceptor specificity modulation in GH29 α-L-fucosidase PsaFuc

GH29 α-L-fucosidases can synthesize valuable fucosylated oligosaccharides via transglycosylation. These α-L-fucosidases display varying acceptor specificities, yet the modulatory mechanism remains poorly understood. Here, we characterized PsaFuc, a GH29 α-L-fucosidase that displays donor-dependent acceptor specificity. With pNP-α-L-fucose as the donor, PsaFuc accepted only fructose as the acceptor with strict acceptor specificity; in contrast, with oNP-β-D-galactose as the donor, it accepted various monosaccharides as acceptors with broad acceptor specificity. Structural and biochemical analyses revealed that the third domain (domain C) of PsaFuc, a C-terminal non-catalytic domain, participates in forming the substrate binding pocket, which exhibits high binding affinity for L-fucose but low binding affinity for D-galactose. Molecular dynamics simulations further suggested that the differential binding affinities for the donor glycosyl moieties (L-fucose vs. D-galactose) influence the conformation of domain C, particularly residue R505, which is involved in acceptor binding. The differential positioning of R505, either close to or away from the catalytic domain, alters the steric restriction of the acceptor binding pocket, thereby modulating acceptor specificity. The designed R505A mutant broadened the acceptor spectrum, confirming the domain C-mediated structural basis underlying donor-dependent acceptor specificity of PsaFuc. This study deepens our understanding of transglycosylation reactions for synthesizing fucosylated oligosaccharides.