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

DOI: 10.1021/jacs.6b11247 DOI Help

Authors: Santiago Alonso-gil (Universitat de Barcelona) , Alexandra Males (The University of York) , Pearl Z. Fernandes (University of Melbourne) , Spencer J. Williams (University of Melbourne) , Gideon J. Davies (University of York) , Carme Rovira (Universitat de Barcelona; Institució Catalana de Recerca i Estudis Avançats (ICREA))
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of The American Chemical Society

State: Published (Approved)
Published: January 2017
Diamond Proposal Number(s): 13587

Abstract: Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B2,5 or 3H4 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 4C1 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 4C1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an OS2 conformation (consistent with catalysis through a B2,5 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-α-mannobiose. This complex revealed unambiguous distortion of the −1 subsite mannoside to an OS2 conformation, matching that predicted by theory and supporting an OS2 → B2,5 → 1S5 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.

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography , I04-Macromolecular Crystallography