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

TY - JOUR

T1 - Computational design-of-experiment unveils the conformational reaction coordinate of GH125 α-mannosidases

AU - Alonso-Gil, Santiago

AU - Males, Alexandra

AU - Fernandes, Pearl

AU - Williams, Spencer J.

AU - Davies, Gideon John

AU - Rovira, Carme

N1 - © 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

PY - 2017/1/25

Y1 - 2017/1/25

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85019057195&partnerID=8YFLogxK

U2 - 10.1021/lacs.6b11247

DO - 10.1021/lacs.6b11247

M3 - Article

SN - 0002-7863

VL - 139

SP - 1085

EP - 1088

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 3

M1 - ja-2016-11247

ER -