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The C-terminal region of xylanase domain in Xyn11A from Paenibacillus curdlanolyticus B-6 plays an important role in structural stability
Authors:Junjarus Sermsathanaswadi  Somsak Pianwanit  Patthra Pason  Rattiya Waeonukul  Chakrit Tachaapaikoon  Khanok Ratanakhanokchai  Krisna Septiningrum  Akihiko Kosugi
Institution:1. Department of Chemical Technology, Faculty of Science and Technology, Suan Dusit Rajabhat University, 295 Rajasrima Rd., Dusit, Bangkok, 10300, Thailand
2. School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkuntien, Bangkok, 10150, Thailand
3. Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki, 305-8686, Japan
4. Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand
5. Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkuntien, Bangkok, 10150, Thailand
6. University of Tsukuba Graduate School of Life and Environmental Sciences, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan
Abstract:Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a major xylanase subunit, designated Xyn11A, which includes two functional domains belonging to glycosyl hydrolase family-11 (GH11) and carbohydrate binding module family-36 (CBM36) and possesses a glycine and asparagine-rich linker (linker). To clarify the roles of each functional domain, recombinant proteins XynXL and XynX (CBM36 deleted and CBM36 and linker deleted, respectively) were constructed. Their xylanase activities were similar toward soluble xylan, whereas XynXL showed decreased hydrolysis activity toward insoluble xylan while XynX had no xylanase activity. To determine the significance of the linker and its neighbor region, XynX was subjected to secondary structural alignments using circular dichroism (CD) spectroscopy and three-dimensional (3D) structural analysis. A seven amino acid (NTITIGG) neighbor linker sequence was highly conserved among GH11 xylanases of Paenibacillus species. Although XynX exhibited a typical GH11 xylanase structure, conformational gaps were observed in the β6- and β12-sheets and in CD spectra. Flipping of the Arg163 side chains in the subsite was also observed upon analysis of superimposed models. Docking analysis using xylohexaose indicated that flipping of the Arg163 side chains markedly affected substrate binding in the subsite. To identify the amino acids related to stabilizing the substrate binding site, XynX with an extended C-terminal region was designed. At least seven amino acids were necessary to recover substrate binding and xylanase activity. These results indicated that the seven amino acid neighbor Xyn11A linker plays an important role in the activity and conformational stability of the xylanase domain.
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