期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 139, 期 3, 页码 1085-1088出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.6b11247
关键词
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资金
- Royal Society
- Biotechnology and Biological Sciences Research Council
- Australian Research Council [FT130100103]
- Spanish Ministry of Economy and Competitiveness [CTQ2014-55174-P]
- GENCAT [2014SGR-987]
- Biotechnology and Biological Sciences Research Council [1644614] Funding Source: researchfish
- BBSRC [1644614] Funding Source: UKRI
- Australian Research Council [FT130100103] Funding Source: Australian Research Council
- ICREA Funding Source: Custom
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.
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