Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides

Abstract

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 α-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 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.

Figures

Fig 1

Temporal glycoprofile of abundant neutral HMO consumption by B. longum subsp. infantis ATCC 15697. HMO composition results are shown for a representative isomer signifying a characteristic oligosaccharide composition. All samples were analyzed at least in duplicate. LNT, lacto-N-tetraose-like composition; LNH, lacto-N-hexaose-like composition; F-LNH, fucosylated lacto-N-hexaose-like composition; DF-LNH, difucosylated lacto-N-hexaose-like composition; F-LNO, fucosylated lacto-N-octaose-like composition; DF-LNO, difucosylated lacto-N-hexaose-like composition.

Fig 2

Phylogenetic relationships of fucosidases encoded by select bacteria. Branch lengths are in the same units (number of amino acid substitutions per site) as those of the evolutionary distances used to construct the tree. The evolutionary history was inferred by the maximum likelihood method, followed by 100 bootstrapped replicates. The organism and loci are listed for those fucosidases found in glycoside hydrolase family 29 (A) and glycoside hydrolase family 95 (B).

Fig 3

ATCC 15697 fucosidase gene expression during carbohydrate fermentation. Gene expression was calculated relative to levels when grown on lactose as the sole carbon source. Averages from three independent experiments are shown, and bars represent standard errors of the means.

Fig 4

Ribbon drawing of the overall structure of Blon_2336. The N-terminal catalytic domain is shown by the various colors, from the blue at the N terminus to red at the C terminus. The β-strands and α-helices that form the (β/α)8 barrel are labeled in black. The C-terminal carbohydrate-binding domain is shown in lime green, with 9 β-strands labeled in magenta. A tyrosine from a possible short peptide at the active site of the catalytic domain (see description in the text) is drawn in stick format. A dashed curved line indicates a missing part in the final structural model (see the text). The N and C termini of Blon_2336 are also labeled.

Fig 5

Structural alignment of the active sites of Blon_2336 and TM0306 in complex with fucose. Blon_2336 is shown in cyan, and TM0306 is shown in green-yellow. All active site residues and the fucose from the TM0306/fucose complex are drawn in stick format. The residues from Blon_2336 and TM0306 are labeled in blue and orange, respectively. Equivalent residues from two molecules are paired together. Some visible strands from the (β/α)8 barrel are labeled in black. The tyrosine shown in Fig. 4 is approximately at the position of the fucose in this figure, and it is not shown for the sake of clarity.