Alonso-Gil, Santiago, Males, Alexandra orcid.org/0000-0002-7250-8300, Fernandes, Pearl et al. (3 more authors) (2017) Computational design-of-experiment unveils the conformational reaction coordinate of GH125 α-mannosidases. Journal of the American Chemical Society. ja-2016-11247. 1085–1088. ISSN 1520-5126
Abstract
Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B 2,5 or 3H 4 transitionstate (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 4 C 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 GH12S active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4 C 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 oS 2 conformation (consistent with catalysis through a B 2,5 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 oS 2 conformation, matching that predicted by theory and supporting an oS 2 → B 2,5 → 1S 5 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 enzymebound species.
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Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2016 American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Chemistry (York) |
Depositing User: | Pure (York) |
Date Deposited: | 04 Jan 2017 09:17 |
Last Modified: | 16 Oct 2024 13:28 |
Published Version: | https://doi.org/10.1021/lacs.6b11247 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1021/lacs.6b11247 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:109931 |
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