Supplementary MaterialsData_Sheet_1

Supplementary MaterialsData_Sheet_1. hand, the GEI of sucrose phosphorylase, a GH13 enzyme, displays the glucose distorted within a 1amylosucrase (glucose, depending on if the catalytic drinking water is normally properly positioned and focused for catalysis or it really is on its way in. Open in a separate window Number 1 sugars changed from 4subsite by QM/MM metadynamics, using the Cremer-Pople puckering coordinates / as collective variables. This is a well-tested approach that we possess used with success to analyse the conformation of carbohydrates in isolation or in the active sites of GHs (Biarns et al., 2007; Alonso-Gil et al., 2017). The computed FEL, demonstrated in Number 2, confirms the most stable conformation is definitely 4sugar for probably the most stable 4sugar (-glucosyl) of -glucosyl residue and the enzyme in the Michaelis complex (energies in kcal/mol, distances in ?). sugars, which collapses to an undistorted 4sugar during the reaction, in relation with the hydrogen relationship between the nucleophile residue (Asp286) and the 6-OH. The development of the sugars conformation during the glycosylation reaction, defined from the puckering coordinate, is definitely shown in Number 6. From your MC to the TS, the conformation evolves from 4sugar (-glucosyl residue) during the glycosylation reaction. Deglycosylation Reaction As demonstrated above, the glycosylation reaction leads to an active site configuration in Y-29794 Tosylate which the sugars is in a 4sugar, Asp393, and the Glu328 catalytic residue. The new configuration, hereafter named GEI*, is definitely separated from the initial GEI by a small free energy barrier (4.7 kcal/mol; Number S5). Amazingly, the sugars at GEI* is definitely preactivated for catalysis, as it exhibits a distorted conformation (sugars conformation along the deglycosylation reaction pathway is definitely shown in Number 5 (bottom panels) and Number 9, whereas Table 3 lists the development of the most important distances. The deglycosylation reaction begins by cleavage of the glycosyl-Asp286 bond, followed by attack of the water molecule on the anomeric carbon, while the water forms a tight hydrogen bond with the acid/base residue (Glu328). The system overcomes the transition state (TS2) and the conformation of the sugar changes to an sugar (-glucosyl residue) during the deglycosylation reaction. Table 3 Calculated values of the most relevant catalytic distances (in ?) and their standard deviations along the deglycosylation minimum energy pathway. subsite adopts a 4sugar adopts a reactive sugar conformation evolved from 4sugar (-glucosyl); (ii) the sucrose, the acid/base residue (Glu328, capped at the C) and the nucleophile residue (Asp286, capped at the C) for the simulation of the glycosylation reaction; (iii) same as (ii) plus the catalytic water, for the simulation of the deglycosylation reaction. In all cases, the frontier atoms between QM and MM region were described using pseudopotential carbon link atoms. The fictitious electronic mass of the Car-Parrinello Lagrangian was taken as 600 au and the timestep was set at 0.12 fs in all CPMD simulations. All systems were enclosed in an isolated cubic box of 12.0 ? 12.0 ? 12.0 ?, using a fictitious electron mass of 700 au and a time step of 0.12 fs. The Kohn-Sham orbitals were expanded in a plane wave (PW) basis set with a kinetic energy cutoff of 70 Ry. Ab initio pseudopotentials generated within the Troullier-Martins scheme were employed (Troullier and Martins, 1991). The Perdew, Burke and Ernzerhoff generalized gradient-corrected approximation (PBE) (Perdew et al., 1996) was selected in view of its good performance in previous work on isolated sugars (Biarns et al., 2007; Marianski et al., 2016), glycosidases (Jin Y-29794 Tosylate et al., 2016) and glycosyltransferases (Bilyard et al., 2018). QM/MM Metadynamics Simulations QM/MM metadynamics (Laio and Parrinello, 2002; Barducci et al., 2011) simulations were performed to characterize the conformational FEL of the -glucosyl residue of PKX1 sucrose in the active site of em Np /em AS and to simulate the different steps of the enzymatic reaction. The following collective variables were used: (i) Conformational FEL: the Cremer-Pople puckering coordinates (Cremer and Pople, 1975) phi and theta (, ) Y-29794 Tosylate of the -glucosyl unit, following the methodology previously used in our group to rationalize and predict catalytic itineraries of GHs (Biarns et al., 2007; Ardvol and Rovira, 2015; Iglesias-Fernandez et al., 2015): (ii) glycosylation reaction: three collective variables representing the proton transfer (CV1), the nucleophilic attack (CV2) and the glycosidic bond cleavage (CV3) (Figure S3); (iii) deglycosylation.