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

Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B-2,B-5 or H-3(4) transition state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-alpha-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a non-hydrolyzable 1,6-alpha-thiomannoside substrate mimic bound across the active site revealed an undistorted C-4(1) 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-4(1) 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-0(2) conformation (consistent with catalysis through a B-2,B-5 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-alpha-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an S-0(2) conformation, matching that predicted by theory and supporting an S-0(2) -> B-2,(5) -> S-1(5) conforinational itinerary for GH125 alpha-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.