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Instruments / Technical Area	Publication Details	Published	Actions
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Computational design of experiment unveils the conformational reaction coordinate of GH125 α-mannosidases Santiago Alonso-Gil , Alexandra Males , Pearl Z. Fernandes , Spencer J. Williams , Gideon J. Davies , Carme Rovira Journal Of The American Chemical Society DOI: 10.1021/jacs.6b11247 Diamond Proposal Number(s): [13587] Open Access Show Abstract ▼ 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.	Jan 2017

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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography

Computational design of experiment unveils the conformational reaction coordinate of GH125 α-mannosidases

Santiago Alonso-Gil , Alexandra Males , Pearl Z. Fernandes , Spencer J. Williams , Gideon J. Davies , Carme Rovira

Journal Of The American Chemical Society

DOI: 10.1021/jacs.6b11247

Diamond Proposal Number(s): [13587]