MM-PBSA free energy analysis of endo-1,4-xylanase II (XynII)–substrate complexes: binding of the reactive sugar in a skew boat and chair conformation

Abstract

The binding of xylotetraose in different conformations to the active site of endo-1,4-β-xylanase II (XynII) from Trichoderma reesei was studied using molecular dynamics (MD) simulations and free energy analyses employing the MM-PBSA (Molecular Mechanics-Poisson–Boltzmann Surface Area) method. MD simulations of 1 ns were done for the substrate xylotetraose having the reactive sugar, which is bound in the −1 subsite of XynII in the ⁴C₁ (chair) and ²S_O (skew boat) ground state conformations, and for the transition state of the XynII catalysed hydrolysis of the β-glycosidic linkage. According to the simulations and free energy analysis, XynII binds the substrate with the −1 sugar in the ²S_O conformation 59.8 kJ mol⁻¹ tighter than the substrate with the sugar in the ⁴C₁ conformation. The reactive ²S_O conformation resembles closely the reaction transition state and has the breaking glycosidic bond in a pseudo-axial orientation ready for facile bond cleavage. The transition state was calculated to be bound 77.1 kJ mol⁻¹ tighter than the ⁴C₁ ground state conformation. The molecular mechanical interaction energy between the enzyme and the reactive pyranoside unit at the −1 subsite was 75.7 kJ mol⁻¹ more favorable for the binding of the ²S_O conformation than the ⁴C₁ conformation, explaining the clearly tighter binding of the reactive structure. The results of this study indicate that in the Michaelis complex XynII, a member of the family 11 xylanase, the substrate is bound in a skew boat conformation and in the catalytic reaction, the −1 sugar proceeds from the ⁴C₁ conformation through ²S_O to the transition state with the −1 sugar in the ^2,5B conformation.