Abstract
The human colonic bacterium Bacteroides thetaiotaomicron, which plays an important role in maintaining human health, produces an extensive array of exo-acting glycoside hydrolases (GH), including 32 family GH2 glycoside hydrolases. Although it is likely that these enzymes enable the organism to utilize dietary and host glycans as major nutrient sources, the biochemical properties of these GH2 glycoside hydrolases are currently unclear. Here we report the biochemical properties and crystal structure of the GH2 B. thetaiotaomicron enzyme BtMan2A. Kinetic analysis demonstrates that BtMan2A is a beta-mannosidase in which substrate binding energy is provided principally by the glycone binding site, whereas aglycone recognition is highly plastic. The three-dimensional structure, determined to a resolution of 1.7 A, reveals a five-domain structure that is globally similar to the Escherichia coli LacZ beta-galactosidase. The catalytic center is housed mainly within a (beta/alpha)8 barrel although the N-terminal domain also contributes to the active site topology. The nature of the substrate-binding residues is quite distinct from other GH2 enzymes of known structure, instead they are similar to other clan GH-A enzymes specific for manno-configured substrates. Mutagenesis studies, informed by the crystal structure, identified a WDW motif in the N-terminal domain that makes a significant contribution to catalytic activity. The observation that this motif is invariant in GH2 mannosidases points to a generic role for these residues in this enzyme class. The identification of GH-A clan and GH2 specific residues in the active site of BtMan2A explains why this enzyme is able to harness substrate binding at the proximal glycone binding site more efficiently than mannan-hydrolyzing glycoside hydrolases in related enzyme families. The catalytic properties of BtMan2A are consistent with the flexible nutrient acquisition displayed by the colonic bacterium.
Highlights
The human colonic bacterium Bacteroides thetaiotaomicron, which plays an important role in maintaining human health, produces an extensive array of exo-acting glycoside hydrolases (GH), including 32 family GH2 glycoside hydrolases
In B. thetaiotaomicron, the vast majority of these enzymes are in glycoside hydrolase families (GHs)3 (4, 5) that contain mainly exo-acting enzymes, which may suggest that the organism is able to utilize the highly complex saccharide decorations appended to the backbone of plant structural polysaccharides, and host proteins presented on the surface of the intestinal mucosa
Trp-395 is equivalent to Trp-137 in CmMan5A, the BtMan2A tryptophan is displaced in the same plane and its indole nitrogen may interact with O-2, rather than O-3 of the substrate
Summary
The human colonic bacterium Bacteroides thetaiotaomicron, which plays an important role in maintaining human health, produces an extensive array of exo-acting glycoside hydrolases (GH), including 32 family GH2 glycoside hydrolases. In B. thetaiotaomicron, the vast majority of these enzymes are in glycoside hydrolase families (GHs) (4, 5) that contain mainly exo-acting enzymes, which may suggest that the organism is able to utilize the highly complex saccharide decorations appended to the backbone of plant structural polysaccharides, and host proteins presented on the surface of the intestinal mucosa. This expansion in exo-GHs is evident in families 2 and 43, in both of which B. thetaiotaomicron has more than 30 members (3). Site-directed mutagenesis in conjunction with sequence comparisons of GH2 enzymes reveals active site residues that confer specificity for terminal mannosides
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