Structure And Function Analysis Of Xylanase Xyn11B’s Catalytic Domain From Sorangium Cellulosum

Sorangium cellulosum can grow under the condition of crystal cellulose (filter paper) as the sole carbon source. Its efficient degradation relies on the contact between cells and the substrate. One epothilone producing strain S. cellulosum So0157-2was isolated previously which can grow using filter paper or xylan as the only carbon source. S. cellulosum So0157-2’s genome contains a series of genes encoding xylanases from different GH family.It is previously found that S. cellulosum So0157-2xylanases have some special characters. During the characterization of Xyn11B-C which includes the catalytic domain of a GH11xylanase, we found that Xyn11B-C possesses certain thermal stability as well as certain substrate degradation pattern. When hydrolyzes xylan, it only produces xylobiose. The latter property has rarely been reported so far.In this study, a further examination was taken to analyze Xyn11B-C’s substrate degradation pattern. The product of Xyn11B-C hydrolyzing PNP-X2and ANTS-Xn was examined respectively. It was found Xyn11B-C hydrolyzes PNP-X2producing PNP and xylobiose and hydrolyzes ANTS-X4, ANTS-X5, and ANTS-X6releasing xylobiose. These results indicate Xyn11B-C degrades substrate releasing xylobiose from the non-reducing end of the sugar chain.Early studies only focused on the amino acid sequence and enzymatic properties of Xyn11B-C and some other xylanases from S. cellulosum So0157-2, but not on substrate degradation mechanism at structural level. To conduct a deeper understanding of Xyn11B-C, we used the protein crystallography techniques to explore the three-dimensional structure of Xyn11B-C.In this study, we constructed the expression vector pGL01-Xyn11B-C. By means of heterologous expression, Ni affinity chromatography, removal of His tag, ion exchange and gel chromatography, Xyn11B-C’s protein sample was prepared which could be used for crystallization. After the screening and optimization of crystallization conditions, we obtained Xyn11B-C crystal and the three-dimensional structure of Xyn11B-C by X-ray diffraction experiment. A Xyn11B-C molecule consists of15β-strands and one a-helix. Similar to other typical GH11xylanases, Xyn11B-C has a right-hand structure with the catalytic site located in the center of the "palm". We also speculated the-1and-2subsite of Xyn11B-C. According to the results of amino acid sequence and structure alignments of Xyn11B-C with some other GH11xylanase, we hypothesized that Xyn11B-C loop L1(residues214-227) and L2(residues316-325) may account for the special property that Xyn11B-C only produced xylobiose.Furthermore, in order to understand how Xyn11B-C combines with its substrate, we have made efforts to get the crystal structure of Xyn11B-C mutant-substrate complex. Xyn11B-C(E331A/Q) were purified and crystallization condition screening and optimization was taken. We have obtained the crystal of Xyn11B-C(E331Q)-X3, Xyn11B-C(E331Q)-X4and Xyn11B-C(E331Q)-X5complex but not their structure yet. Only the crystal structure of Xyn11B-C(E331Q) itself was analyzed.In summary, this paper conducted further analysis of the way Xyn11B-C degrading xylan based on previous Xyn11B-C enzymatic study. By means of protein crystallography, we obtained the structures of Xyn11B-C and Xyn11B-C(E331Q) and predicted the way Xyn11B-C binded with the substrate.We supposed the reason Xyn11B-C having this relatively special enzymatic property at its structural level and revealed the relationship of Xyn11B catalytic domain’s amino acid sequence, biochemical properties and its protein structure.