Endo-1,4-β-xylanase (EC 3.2.1.8) is the enzyme from Ruminococcus albus 8 (R.albus 8) (Xyn10A), and catalyzes the degradation of arabinoxylan, which is a major cell wall non-starch polysaccharide of cereals.The crystallographic structure of Xyn 10A is still unknown.For this reason, we report a computer-assisted homology study conducted to build its three-dimensional structure based on the known sequence of amino acids of this enzyme.In this study, the best similarity was found with the Clostridium thermocellum (C.thermocellum) N-terminal endo-1,4-β-D-xylanase 10 b.Following the 100 ns molecular dynamics (MD) simulation, a reliable model was obtained for further studies.Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods were used for the substrate xylotetraose having the reactive sugar, which was bound in the-1 subsite of Xyn10A in the 4C1 (chair) and 2So (skew boat) ground state conformations.According to the simulations and free energy analysis, Xyn10A binds the substrate with the-1 sugar in the 2So conformation 39.27 kcal·mol-1 tighter than the substrate with the sugar in the 4C1 conformation.According to the Xyn1 0A-2So Xylotetraose (X4(sb) interaction energies, the most important subsite for the substrate binding is subsite-1.The results of this study indicate that the substrate is bound in a skew boat conformation with Xyn10A and the -1 sugar subsite proceeds from the 4C1 conformation through 2So to the transition state.MM-PBSA free energy analysis indicates that Asn187 and Trp344 in subsite-1 may an important residue for substrate binding.Our findings provide fundamental knowledge that may contribute to further enhancement of enzyme performance through molecular engineering.