Abstract
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 Xyn10A 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 Xyn10A-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.
Highlights
Ruminococcus albus 8 (R. albus 8) is one of the most actively fibrolytic ruminal bacteria in the world, which can degrade cellulose and hemicellulose in forages such as alfalfa and grass hays [1,2,3].It is well known that R. albus 8 has a wide range of protein activities [4,5,6].Xylans are abundant hemicellulolytic components of the plant cell wall, which can be degraded into the corresponding oligomeric and monomeric sugars, providing a major source of renewable energy.The main chain of xylan is composed of 1,4-D-xylose subunits, which is usually decorated with various side chain residues of 1,2-α-D-glucuronic acid, or its 4-O-methyl ethers, 1,3-α-L-arabinose, and/or O-acetyl groups in the 2 and 3 positions
Xyn10A clusters together with C. thermocellum N-terminal endo-1,4-β-D-xylanase 10 b (Xyn10b) (PDB Id 2W5F) [19]. This suggests that Xyn10A and C. thermocellum N-terminal endo-1,4-β-D-xylanase 10 b (Xyn10b) will form a new clade in GH10 family
To perform homology modeling for parts of the structure conserved among XynA with known crystal structures, previous target-template sequence alignment was performed using the Blast algorithm which gave the highest sequence similarity, 39%, to the C. thermocellum N-terminal endo-1,4-β-D-xylanase 10b (Xyn10b) CBM22-1-GH10 sequences [19]
Summary
Ruminococcus albus 8 (R. albus 8) is one of the most actively fibrolytic ruminal bacteria in the world, which can degrade cellulose and hemicellulose in forages such as alfalfa and grass hays [1,2,3]. Several xylanolytic enzymes are required to release the substituents and sugars from the various xylans, including endo-1,4-β-xylanases (EC 3.2.1.8) [7], acetyl xylan esterases (EC 3.1.1.72) [8], feruloyl esterases (EC 3.1.1.73) [9], α-L-arabinofuranosidases (EC 3.2.1.55) [10], α-glucuronidases (EC 3.2.1.139) [11], and β-D-xylosidase (EC 3.2.1.37) [12]. Inverting glycosidases appear to use a mechanism in which a general acid/base catalyzed direct displacement occurs at the anomeric center through an oxocarbenium ion-like transition state [14,15]. Use a double-displacement mechanism in which a covalent glycosyl-enzyme intermediate is formed and hydrolyzed in a general acid/base catalyzed process through oxocarbenium ion-like transition states, or possibly through oxocarbenium ion intermediates [16,17]. The 3D structure of Xyn10A was built and used to predict the binding pose between the enzyme and ligands
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