Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.

An 1,4-α-Glucosyltransferase Defines a New Maltodextrin Catabolism Scheme in Lactobacillus acidophilus.

The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic α-amylase of family 13, subfamily 20 (LaGH13_20), a maltose phosphorylase of GH65 (LaGH65), and a family 13, subfamily 31, member (LaGH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that LaGH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS with a degree of polymerization of ≥2, with a preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus revealed that the substrate preference of LaGH13_31B toward maltotriose complements the ~40-fold lower kcat of LaGH13_20 toward this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose. The concerted action of LaGH13_20 and LaGH13_31B confers the efficient conversion of MOS to maltose that is phosphorolyzed by LaGH65. Structural analyses revealed the presence of a flexible elongated loop that is unique for a previously unexplored clade of GH13_31, represented by LaGH13_31B. The identified loop insertion harbors a conserved aromatic residue that modulates the activity and substrate affinity of the enzyme, thereby offering a functional signature of this clade, which segregates from 1,6-α-glucosidases and sucrose isomerases previously described within GH13_31. Genomic analyses revealed that the LaGH13_31B gene is conserved in the MOS utilization loci of lactobacilli, including acidophilus cluster members that dominate the human small intestine.IMPORTANCE The degradation of starch in the small intestine generates short linear and branched α-glucans. The latter are poorly digestible by humans, rendering them available to the gut microbiota, e.g., lactobacilli adapted to the small intestine and considered beneficial to health. This study unveils a previously unknown scheme of maltooligosaccharide (MOS) catabolism via the concerted activity of an 1,4-α-glucosyltransferase together with a classical hydrolase and a phosphorylase. The intriguing involvement of a glucosyltransferase likely allows the fine-tuning of the regulation of MOS catabolism for optimal harnessing of this key metabolic resource in the human small intestine. The study extends the suite of specificities that have been identified in GH13_31 and highlights amino acid signatures underpinning the evolution of 1,4-α-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli.