Mechanistic insights into a Ca2+-dependent family of alpha-mannosidases in a human gut symbiont

Abstract

Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are alpha-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca(2+)-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca(2+) plays a key catalytic role in helping distort the mannoside away from its ground-state (4)C(1) chair conformation toward the transition state.

Figure 1. Human N-glycans and the role of α-mannosidases

(a) Illustration of a Man₉(GlcNac)₂ N-glycan with the stereochemistry of the linkages indicated. (b) The enzymatic hydrolysis of α-1,2-mannosides, as catalyzed here by many of the GH92 α-mannosidases.

Figure 2. Three-dimensional structure of GH92 α-1,2-mannosidase Bt3990

(a) Protein cartoon of the Bt3990 structure, in divergent (“wall-eyed”) stereo with the N-terminal domain in red and the C-terminal domain in blue, and with the thiomannobioside (4) substrate mimic shown in ball-and-stick. (b) The solvent-accessible surface of Bt3990 (colored as in a), highlighting how the thiomannobioside substrate mimic (with gray surface) nestles at the interface. (c–e) The maximum-likelihood/σ_A weighted 2F_obs − F_calc densities (contoured at 1σ) for mannoimidazole (c), swainsonine (3) (d) and kifunensine (5) (e). The critical Ca²⁺ ion is shown as a green sphere.

Figure 3. The active center of Bt3990 and catalysis with inversion of anomeric configuration

(a) Schematic diagram of the interactions of Bt3990 with Ca²⁺/mannoimidazole (6) highlighting the interactions of the −1 subsite and the distortion of the ligand toward a B_2,5 conformation. (b) Putative reaction mechanism for an inverting GH92 α-mannosidase in which Asp644 acts as catalytic base, Ca²⁺ bridges the O2 and O3 hydroxyls while also interacting with the attacking water, and Glu533 provides brønsted acid assistance to leaving group departure.

Figure 4. Interactions of thiomannobioside with GH92 α-1,2-mannosidase Bt3990

(a) Structure of the Bt3990 complex cocrystallized with thiomannobioside, with the acid (Glu533), the putative base (Asp644) and the α-1,2 signature motifs (Trp88, His584 and Glu585) shown. The main chain is colored red for the N-terminal domain and blue for the C-terminal domain, showing how the active center is cradled by residues from both domains. (b) Schematic diagram showing the interactions of the aforementioned residues with the thiomannobioside. (c) Structure of the Bt3990 complex, soaked with thiomannobioside (green protein, gray ligand) overlaid with the Bt3990 mannoimidazole (6) complex (yellow). a and c include the maximum-likelihood/σ_A weighted 2F_obs − F_calc density contoured at 1σ.