Computational Design of Experiment Unveils the Conformational Reaction Coordinate of GH125 α-Mannosidases

Abstract

Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B or H transition-state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted C conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH125 active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative C conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an S conformation (consistent with catalysis through a B TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an S conformation, matching that predicted by theory and supporting an S → B → S conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzyme-bound species.

Distortion of mannoimidazole supports a B2,5 boat transition state for the family GH125 α-1,6-mannosidase from Clostridium perfringens

“…18 Instead, a computational approach involving ab initio QM/MM metadynamics was used to model the conformation of substrate, 1,6-α-mannobiose, in the enzyme active site, by in silico substitution of the sulfur in the experimentally determined X-ray structure. 19 This calculation predicted that the O-glycoside favoured an O S 2 conformation on-enzyme and hinted at an O S 2 → B 2,5 ‡ → 1 S 5 conformational itinerary, which was fully supported by QM/MM simulations of the reaction mechanism (Fig. 2b).…”

“…2c). 23 However, analysis of published complexes with GH125 enzymes shows that the general acid residue is located below the mean plane of the ring, 18,19 suggesting that there are poor prospects for binding of MIm to CpGH125. Yet, there are now several examples of glycoimidazoles binding to glycosidases with good affinity even without achieving a prototypical interaction between the enzymatic general acid and the imidazole nitrogen, such as β-glucosidases of family GH116, 24 α-mannanases of family GH99 25 and α-mannosidases of family GH47 10 (Fig.…”

“…Binding stoichiometry of >1 for basic sugar-shaped heterocycles is unusual and suggests that the ligand may not solely be binding at the −1 subsite. Previous work has identified three subsites, −1, +1 and +2, in CpGH125 through a complex with 1,6-α-mannotriose (PDB ID: 5M7Y), 19 and studies of a di-saccharide substrate that binds in the −1/+1 subsites revealed saturation kinetics, whereas a monosaccharide substrate, 2,4dinitrophenyl α-mannoside (DNPMan), that bound only in the −1 subsite did not. 18,26 Previous poor electron density in the +2 subsite suggests that this may represent the low affinity binding site, a conclusion supported by subsequent X-ray crystallographic studies (vide infra).…”

“…Direct experimental support for this mechanism was obtained through determination of the Michaelis complex for the catalytically-disabled acid mutant (D220N) of CpGH125 in complex with 1,6-α-mannobiose, which revealed an O S 2 conformation consistent with the computational predictions. 19 As the ultimate determinant of the activation barrier for catalysis, the TS is the cardinal feature of the conformational itinerary. Mannoimidazole (3) (MIm) has emerged as a powerful chemical probe for TS conformation of mannosidases owing to its flattened nature, with an sp 2 -hybridized anomeric centre and energetically feasible access to all of the possible stereoelectronically-compliant conformations expected for an oxocarbenium ion-like transition state (Fig.…”

Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach

“… Conformation itineraries for inverting α‐mannosidases proceeding through left: O S 2 →[ B 2,5 ] ≠ → 1 S 5 (e.g., GH125) and right: 3 S 1 →[ 3 H 4 ] ≠ → 1 C 4 (GH47) pathways. Catalytic residues acting as proton donors/acceptors are omitted for clarity.…”