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Insights into the roles of non-catalytic residues in the active site of a GH10 xylanase with activity on cellulose

DOI: 10.1074/jbc.M117.807768 DOI Help

Authors: Yindi Chu (Feed Research Institute, China) , Tao Tu (Feed Research Institute, China) , Leena Penttinen (University of Eastern Finland) , Xianli Xue (Feed Research Institute, China) , Xiaoyu Wang (Feed Research Institute, China) , Zhuolin Yi (Chengdu Institute of Biology, Chinese Academy of Sciences) , Li Gong (College of Animal Science, China) , Juha Rouvinen (University of Eastern Finland) , Huiying Luo (Feed Research Institute, China) , Nina Hakulinen (University of Eastern Finland) , Bin Yao (Feed Research Institute, China) , Xiaoyun Su (Feed Research Institute, China)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: October 2017

Abstract: Bifunctional glycoside hydrolases have potential for cost saving in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals. The N-terminal GH10 domain of a bifunctional multimodular enzyme CbXyn10C/Cel48B from Caldicellulosiruptor bescii, is an enzyme able to degrade xylan and cellulose simultaneously. However, the molecular mechanism underlying its substrate promiscuity has not been elucidated. Herein, we discovered that the binding cleft of CbXyn10C would have at least six sugar binding subsites by using isothermal titration calorimetry analysis of the inactive E140Q/E248Q mutant with xylo- and cellooligosaccharides. This was confirmed by determining the catalytic efficiency of the wild-type enzyme on these oligosaccharides. The free form and complex structures of CbXyn10C with xylose- or glucose-configured oligosaccharide ligands were further obtained by crystallographic analysis and molecular modeling and docking. CbXyn10C was found to have a typical (β/α)8-TIM barrel fold and "salad-bowl" shape of GH10 enzymes. In complex structure with xylo-oligosaccharides, seven sugar-binding subsites were found and many residues responsible for substrate interactions were identified. Site-directed mutagenesis indicated that six and ten amino acid residues were key residues for xylan and cellulose hydrolysis, respectively. The most important residues are centered on the subsites -2 and -1 near the cleavage site, while residues playing moderate roles could be located at more distal regions of the binding cleft. Manipulating the residues directly or indirectly interacting with substrates in the distal regions improved the activity of CbXyn10C on xylan and cellulose. Most of the key residues for cellulase activity are conserved across GH10 xylanases. Revisiting randomly selected GH10 enzymes revealed unreported cellulase activity, indicating that the dual function may be a more common phenomenon than has been expected.

Journal Keywords: cellulase; crystal structure; glycoside hydrolase; molecular docking; mutagenesis; Bifunctional; Biofuels; GH10; Xylanase

Diamond Keywords: Biofuel; Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Energy, Chemistry

Instruments: I04-Macromolecular Crystallography

Other Facilities: ESRF

Added On: 20/11/2017 12:03

Discipline Tags:

Bioenergy Earth Sciences & Environment Biotechnology Sustainable Energy Systems Energy Climate Change Biochemistry Catalysis Chemistry Structural biology Engineering & Technology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)