Bioinformatics-aided function exploration of GH29 fucosidases from human gut Parabacteroides.

Abstract

Gut microbes produce α-l-fucosidases critical for utilizing human milk oligosaccharides, mucosal and dietary glycans. Although gut Parabacteroides have garnered attention for their impact on host health and disease, their CAZymes remain poorly studied. CAZome analysis of eleven gut Parabacteroides type strains revealed their capacity to degrade mucin O-glycans. Their abundance of GH29 fucosidases caught our attention, and we predicted the functional profiles of 46 GH29 fucosidases using in silico approaches. Our findings showed diverse linkages specificities and species-specific distributions, with over half of GH29 enzymes functioning as α1,3/4 fucosidases, essential for acting on Lewis antigen epitopes of mucin O-glycans. We further enzymatically validated 4 novel GH29 sequences from poorly characterized groups. PgoldGH29A (cluster37 GH29BERT, GH29:75.1 CUPP) does not act on tested natural substrates. PgoldGH29B (cluster1 GH29BERT, GH29:84.1 CUPP) functions as a strict α1,3/4 fucosidase. PgoldGH29C (cluster14 GH29BERT, GH29:29.1 CUPP) displays unprecedented substrate specificity for α1,2/3/4 disaccharides. PgoldGH29D (cluster4 GH29BERT, GH29:6.2 CUPP) acts on α1,2/3/4/6 linkages similar to enzymes from GH29:6.1 CUPP but prefers disaccharides over trisaccharides. These results suggest that PgoldGH29B and PgoldGH29D can contribute to mucin O-glycan degradation via their α1,3/4 and α1,2 fucosidase activity, respectively, while the natural substrates of PgoldGH29A and PgoldGH29C may be irrelevant to host-glycans. These insights enhance our understanding of the ecological niches inhabited by gut Parabacteroides and may guide similar exploration in other intriguing gut microbial species.