5M7Y

Crystal structure of GH125 1,6-alpha-mannosidase mutant from Clostridium perfringens in complex with 1,6-alpha-mannotriose

Summary for 5M7Y

• Entry DOI: 10.2210/pdb5m7y/pdb
• Related: 5M7I
• Descriptor: 1,6-alpha-mannosidase, alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose, MAGNESIUM ION, ... (4 entities in total)
• Functional Keywords: glycoside hydrolase, mannosidase, carbohydrate, hydrolase
• Biological source: Clostridium perfringens (strain 13 / Type A)
• Total number of polymer chains: 1
• Total formula weight: 51414.06

Authors

Males, A.,Alonso-Gil, S.,Fernandes, P.,Williams, S.J.,Rovira, C.,Davies, G.J. (deposition date: 2016-10-28, release date: 2016-11-30, Last modification date: 2024-05-08)

Primary citation

Alonso-Gil, S.,Males, A.,Fernandes, P.Z.,Williams, S.J.,Davies, G.J.,Rovira, C.
Computational Design of Experiment Unveils the Conformational Reaction Coordinate of GH125 alpha-Mannosidases.
J. Am. Chem. Soc., 139:1085-1088, 2017

PubMed Abstract: Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B or H 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 C 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 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 conformation (consistent with catalysis through a B 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 S conformation, matching that predicted by theory and supporting an S → B → S 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.

PubMed: 28026180
DOI: 10.1021/jacs.6b11247

Experimental method

X-RAY DIFFRACTION (1.55 Å)