Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α- l -Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

Abstract

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the ␣-(132) linkage of 2-fucosyllactose, and a gene encoding 1,2-␣-L-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal ␣-(132)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2-fucosyllactose was determined to be inversion by using 1 H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).␣-L-Fucosyl residues are frequently found at the nonreducing termini of various glycoconjugates, including blood group substances, milk oligosaccharides, gastric and submaxillary mucins, and serum glycoproteins (30, 37). The results of recent studies indicate that such terminal fucosyl residues attached by

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.…”

Structural and Thermodynamic Analyses of Solute-binding Protein from Bifidobacterium longum Specific for Core 1 Disaccharide and Lacto-N-biose I

“…B. longum JCM1217 has endo-␣-N-acetylgalactosaminidase (BL0464 homolog), which specifically cleaves the GNB unit (core 1 sugar) from mucin (74). Moreover, extracellular 1,2-␣-L-fucosidase is found in Bifidobacterium bifidum (75). HMO and mucin glycans may be degraded by extracellular glycosidases into disaccharide units, such as LNB, GNB, lactose, and LacNAc, before incorporation by specific transporters.…”

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.…”