title: Computational Design of Experiment Unveils the Conformational Reaction Coordinate of GH125 α-Mannosidases.
abstract: Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B2,5or3H4transition-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 undistorted4C1conformation 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 uninformative4C1conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in anOS2conformation (consistent with catalysis through a B2,5TS). 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 anOS2conformation, matching that predicted by theory and supporting anOS2→ B2,5→1S5conformational 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.
authors.affiliation: Departament de Química Inorgànica i Orgànica (Secció de Química Orgànica) & Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona , 08028 Barcelona, Spain.
authors.forename: Santiago
authors.initials: S
authors.lastname: Alonso-Gil
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York , YO10 5DD York, United Kingdom.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Males
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Melbourne, Victoria 3010, Australia.
authors.forename: Pearl Z
authors.initials: PZ
authors.lastname: Fernandes
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Melbourne, Victoria 3010, Australia.
authors.forename: Spencer J
authors.identifier: 0000-0001-6341-4364
authors.initials: SJ
authors.lastname: Williams
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York , YO10 5DD York, United Kingdom.
authors.forename: Gideon J
authors.initials: GJ
authors.lastname: Davies
authors.affiliation: Departament de Química Inorgànica i Orgànica (Secció de Química Orgànica) & Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona , 08028 Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats (ICREA) , 08010 Barcelona, Spain.
authors.forename: Carme
authors.identifier: 0000-0003-1477-5010
authors.initials: C
authors.lastname: Rovira
mesh_terms: D003016:Clostridium perfringens / Q000201:enzymology; D008358:Mannose / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D011487:Protein Conformation; D011789:Quantum Theory; D043323:alpha-Mannosidase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D043323:alpha-Mannosidase; D008358:Mannose