Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 ␣-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 ␣-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.T he genus Bifidobacterium is frequently overrepresented in the breast-fed infant colon relative to its appearance in adults, where these organisms are believed to benefit their host through nutrient supplementation, participating in host energy cycling and binding to preferred host receptor molecules otherwise available to pathogens (12). Selective growth of bifidobacteria has been attributed to utilization of oligosaccharides abundant in human milk (10 to 20 g/liter) that present complex structures resistant to infant digestion (17, 35). Approximately 200 species of human milk oligosaccharides (HMOs) have been characterized that are composed of glucose, galactose, N-acetylglucosamine, and often fucose and/or sialic acid residues via several glycosidic linkages (25). The HMO core is typically elongated from a lactosyl reducing end (Gal1-4Glc) that is linked via 1-3 (or 1-6 in branched molecules) to serial lacto-N-biose I units (Gal1-3GlcNAc) or lactosamine (Gal1-4GlcNAc) with a degree of polymerization of Ն4.As with other fucosylated glycoconjugates, ␣1-2/3/4 fucosyl moieties often shield HMOs from digestion unless this linkage at the nonreducing terminus is first cleaved. Similarly, acidic HMOs or milk sialyloligosaccharides (MSOs) obstruct enzymatic degradation with sialyl residues via ␣2-3/6 linkages. Removal of these termini is postulated to initiate bacterial catabolism of HMOs (2,8,30). To this end, it has been recently demonstrated that Bifidobacterium longum subsp. infantis ATCC 15697 utilizes milk sialyloligosaccharides via a sialidase encoded within a large gene cluster dedicated to HMO metabolism (30).Previous research conducted on bifidobacterial metabolism of fucosylated oligosaccharides identified a Bifidobacterium bifidum ␣1-2-L-fucosidase that exhibited an atypical inverting mechanism (glycoside hydrolase [GH] family 95), termed AfcA (10,22). Inverting glycoside hydrolases modify anomeric stereochemistry via a single nucleophilic displacement, mechanistically contrasting with retaining enzymes, which maintain the anomeric configuration through catalysis of ...