Two distinct  -L-fucosidases from Bifidobacterium bifidum are essential for the utilization of fucosylated milk oligosaccharides and glycoconjugates

Abstract

Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.

Genome analysis of Bifidobacterium bifidum PRL2010 reveals metabolic pathways for host-derived glycan foraging

“…The B. bifidum PRL2010 genome encodes various glycosyl hydrolases putatively implicated in degradation of mucinderived oligosaccharides, including a predicted cell wall-anchored endo-α-N-acetylgalactosaminidase (BBPR_0264), an enzyme that has been shown previously to catalyze the hydrolysis of the O-glycosidic α-linkage between GalNAc and serine/threonine residues of various mucin-type glycoproteins (30)(31)(32). Moreover, the genome of B. bifidum PRL2010 encodes a putative 1,2-α-Lfucosidase (BBPR_0193), as well as a predicted 1,3/4-α-L-fucosidase (BBPR_1360), which releases various α-linked L-fucoses from the oligosaccharide core of the mucin structure (33)(34)(35). Both fucosidases contain a signal peptide, but only BBPR_0193 contains an LPXTG motif, suggesting that this enzyme is secreted and anchored to the cell wall, whereas the presumed fucosidase encoded by BBPR_1360 contains two transmembrane domains, indicating that it is bound to the cell membrane.…”

From food to cell: nutrient exploitation strategies of enteropathogens

“…Bifidobacterium bifidum (Ashida et al, 2009;Katayama et al, 2004). The breakdown of HMOs to SCFAs lowers the gut pH and thus diminishes the growth of harmful bacteria.…”

Unique Sugar Metabolic Pathways of Bifidobacteria

“…54) AfcB consists of 1,493 amino acids and contains an N-terminal signal sequence, a GH29 -L-fucosidase domain, a carbohydrate-binding module (CBM) family 32 domain, a Found In Various ARchitectures (FIVAR) domain, and a C-terminal transmembrane region. The CBM32 domain generally binds a galactose residue at the non-reducing end, 55) which is frequently present in HMO.…”