The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

• Published in: Journal of biotechnology 2013-12, Vol.168 (4), p.403-408
• Main Authors: Khan, Muhammad Imran M., Sajjad, Muhammad, Sadaf, Saima, Zafar, Rehan, Niazi, Umer H.K., Akhtar, Muhammad Waheed
• Format: Article
• Language: English
• Subjects: active sites; Activity enhancement; Binding; Binding Sites; biotechnology; C. thermocellum; carbohydrate binding; Carbohydrate binding module; Carbohydrates; Carbohydrates - chemistry; Catalysis; Catalysts; catalytic activity; Catalytic Domain; cellulosome; Cloning, Molecular; Clostridium thermocellum; Clostridium thermocellum - enzymology; Clostridium thermocellum - growth & development; Endo-1,4-beta Xylanases - chemistry; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - metabolism; Escherichia coli; Esterases; feruloyl esterase; Gene Expression Regulation, Bacterial; Modules; Molecular Docking Simulation; molecular models; Protein Structure, Tertiary; proteins; Substrate Specificity; Thermostability; xylan; Xylanase; Xylanase Z; xylanases
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2013.09.010

•Variants of xynlanase Z of Clostridium thermocellum in combination with CBM6 and CBM22 were expressed in E. coli.•The construct with CBM22 showed 5-fold higher activity as compared to the one containing CBM6.•Location of the binding site inside a tunnel like structure of the CBM6 construct seemed to restrict the substrate binding.•The construct with CBM22 produced a more open structure, facilitating substrate binding and release of the product. Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B′C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B′C were 4200, 4180 and 20,700UμM−1 against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B′C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B′C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.