Fucoidan and xyloglucan hydrolysis predicted by protein domain architecture in GH29 α-l-fucosidases

Abstract Family GH29 α- l -fucosidases hydrolyze fucosylated glycoconjugates via a retaining mechanism and are therefore considered useful for the synthesis of medically relevant molecules such as α-fucosylated human milk oligosaccharides by transfucosylation using naturally abundant α- l -fucosyl donors. Fucoidan and fucosylated xyloglucan are naturally abundant polysaccharides that can serve as α- l -fucosyl donors. However, few GH29 α- l -fucosidases have been reported to hydrolyze these polysaccharides or use them in transfucosylation, although large-scale efforts have been made to characterize members of this family. Here, we describe a domain-context-based analysis that we employed to predict GH29 α- l -fucosidases likely to hydrolyze fucoidan and fucosylated xyloglucan. We predicted fucoidan- and fucosylated xyloglucan-hydrolyzing α- l -fucosidases based on the presence of co-occurring sulfatase domains, and relevant carbohydrate-binding modules and glycoside hydrolase domains, and fine-tuned our predictions using ecological niche information and Conserved Unique Peptide Patterns grouping. We recombinantly expressed and biochemically characterized seven of these predicted GH29 members from Rhodopirellula sp. SWK7 ( Rs Fuc), Rhodopirellula pilleata ( Rp Fuc), Novipirellula artificiosorum ( Na Fuc), Clostridium methylpentosum ( Cm Fuc), Rhodopirellula maiorica ( Rm Fuc), Bacteroides sp. ( Bs Fuc), and Bacterium HR17/ Candidatus Fervidibacter japonicus (HR17Fuc), and demonstrated that they catalyze the hydrolysis of fucoidan and fucosylated xyloglucan. While these α- l -fucosidases did not score positive in a cursory transfucosylation screen, these and other GH29 members predicted to hydrolyze fucoidan or fucosylated xyloglucan may be further explored for their transfucosylation potential in future studies. The domain-context-based prediction of substrate preference described here can be extended for other substrates and to other carbohydrate-active enzyme families. Key points • GH29 alpha-L-fucosidases with fucoidan or xyloglucan-specific domains were predicted • RsFuc, RpFuc, NaFuc, CmFuc, RmFuc, BsFuc, and HR17Fuc were recombinantly expressed • Fucoidan and fucosylated xyloglucan were hydrolyzed by these alpha-L-fucosidases