Structural Analysis Of β-Xylosidase (XylC) From Thermoanaerobacterium Saccharolyticum JW/SL-YS48

The world’s demand for resources and energy is greatly growing, thus much attention has been paid to exploit and utilize renewable resources. The hemicellulose could be a material for renewable resources if effective digestion and transformation of the waste can be achieved. Hemicellulose includes all kinds of heteropolysaccharide, and xylan is one of the major components, which need a series of enzymes to degrade completely. The function of P-xylosidase is to hydrolyze xylooligosaccharide and xylobiose, therefore it has an important role for the xylan degradation. Accordingly, the researches on P-xylosidase structure and function are important.The P-xylosidase we studied comes from Thermoanaerobacterium saccharolyticum JW/SL-YS485, and is named as XylC. XylC contains638amine acid residues, and the molecular weight is about72.6kDa. XylC was purified by heat treatment, DEAE-sepharose, Gel filtration and Hydrophobic interaction, the purity of protein is higher than95%and is suitable for crystallization. Well-diffracting crystals were obtained by sitting-drop vapor diffusion method. The crystals belong to the monoclinic space group P21and collect to2.2A resolution with unit cell parameters a=88.36A, b=202.20A, c=99.87A, p=99.04°. The datasets of mercury-containing derivatives of XylC were collected successfully, and the resolution is better than2.5A. The phase of XylC was solved by the multiple isomorphous replacement (MIR) methodwith SOLVE/RESOLVE software. We used ARP/wARP to build the initial model, following to refine structure by CNS and Refmac5. Afterward, we also determined the complex structures of XylC-xylobiose and XylC-xylose.XylC forms a tetramer in asymmetric unit. Each monomer of P-xylosidase comprises three distinct domains:N-terminal domain (1-204), catalytic domain (205-484) and C-terminal domain (485-638). The architecture of the catalytic domain is right-handed parellel β helix and displays eight complete coils. From the complex structures of XylC-xylobiose and XylC-xylose, the active site possess an opened cavity consisting of amino acid residues E405, D382, K358, E353, W383and W113that are important for enzyme activities. The two catalytic residues, E405(nucleophile) and D382(general acid/base), were determined depending on complex structures. In addition, we proposed that XylC cleaves the glycosidic bond via the retaining mechanism. Seven or eight xylose molecules were observed in one monomer of XylC-xylose complex structure suggesting that XylC has low product inhibition because of non-specific binding mode.In this study, we determine the first crystal structure of β-xylosidase of glycoside hydrolase120family that may give an insight into the structureal and functional features of other GH120family enzymes and may also benefit for further applications.