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. 2015 Feb 1;6(2):1167-1177.
doi: 10.1039/c4sc02240h. Epub 2014 Oct 27.

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases

Javier Iglesias-Fernández  1 , Lluís Raich  1 , Albert Ardèvol  2 , Carme Rovira  1   3
Affiliations

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases

Javier Iglesias-Fernández et al. Chem Sci. .

Abstract

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer-Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2SO → [2,5B]ǂ → 5S1 and 1S3 → [4H3]ǂ → 4C1, which clearly appear in low energy regions of the FEL. Consistently, 2SO and 1S3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the OE → [OS2]ǂ → B2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed OE conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases.

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Figures

Fig. 1
Fig. 1. Cremer and Pople puckering coordinates of a six-membered ring (Q, θ, and φ) and their projection in the x, y plane (q x and q y).
Fig. 2
Fig. 2. Main conformations on the Cremer–Pople sphere along with two of their most used representations: Stoddart (Northern projection) and Mercator. Proposed itineraries for β-xylanases belonging to several GH families are depicted on Stoddart diagram.
Fig. 3
Fig. 3. (a) Computed free energy landscape of cyclohexane resulting from the first principles metadynamics using θ and φ as collective variables (Mercator representation). The continuous lines, separated by 30° in φ, indicate regions corresponding to different canonical conformations. (b) Computed free energy landscape of cyclohexane resulting from the ab initio metadynamics using q x and q y as collective variables (Stoddart representation). Energy values are given in kcal mol–1 and each contour line of the diagram corresponds to 1 kcal mol–1.
Fig. 4
Fig. 4. Probability distribution for the radial coordinate Q in cyclohexane.
Fig. 5
Fig. 5. Computed free energy landscape of β-xylose (Mercator representation) with respect to ring distortion. Energy values are given in kcal mol–1 and each contour line of the diagram corresponds to 1 kcal mol–1. The conformations found in experimental structures of retaining β-xylosidases are represented by red color stars (Michaelis complex structures) and blue color stars (covalent intermediate structures). The conformation of the TS-like inhibitor xylobio-imidazole in complex of family 10 xylanase Cex from Cellulomonas fimi has also been indicated (green color star). For the sake of clarity, only one star is displayed for several structures with nearly identical conformations.
Fig. 6
Fig. 6. Conformational free energy landscapes (Stoddart representation of the puckering sphere Northern hemisphere) obtained for β-xylose (this work), β-glucose (ref. 43) and β-mannose (ref. 48). Each contour line of the diagram corresponds to 1 kcal mol–1 (β-xylose) and 0.5 kcal mol–1 (β-glucose and β-mannose).
Fig. 7
Fig. 7. Results of the MD simulations of Michaelis complex of Trichoderma ressei GH11 xylanase. The graphs represent the distribution of conformations for each simulation. (a) Mutant enzyme. (b) Wild type enzyme.
Fig. 8
Fig. 8. Comparison between OS2 conformation of β-xylose, β-glucose and β-mannose (only one of the possible rotameric states of the exocyclic groups is shown).
Fig. 9
Fig. 9. Variation of the values of the preactivation index ξ as a function of the ring conformation.
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