Research On Catalytic Mechanism Of The Multimodular Glycoside Hydrolase CbXyn10C/Ce148B From Caldicellulosiruptor Bescii

Plant cell walls are comprised of cellulose,hemicellulose and other polymers that are intertwined.Bifunctional or multi-funcational glycoside hydrolases present a means towards cost saving in destruction of plant cell wall polysaccharides for biofuels and bio-based chemicals.CbXyn 10C/Ce148B,from thermophilic anaerobe Caldicellulosiruptor bescii,is a multi-modular protein,with its N-terminus and C-terminus annotated as xylanase and cellulase respectively.As the N-terminus of a bifunctional multimodular enzyme CbXyn10C/Ce148B from Caldicellulosiruptor bescii,the GH10 CbXyn10C is such 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 has no less than six sugar binding subsites by isothermal titration calorimetry analysis of the inactive E140Q/E248Q mutant with xylo-and cellooligosaccharides with increasing degrees of polymerization.This was confirmed by determining the catalytic efficiency of the wild-type enzyme on these oligosaccharides.The apo-form and complex structures of CbXyn10C with xylose-or glucose-configured oligosaccharide ligands were further obtained by crystallography analysis or molecular modeling and docking.CbXyn10C is thus found to have a typical(?/?)8-TIM barrel fold and "salad-bowl" shape of GH10 enzymes.It has seven sugar-binding subsites and many residues are predicted to be responsible for substrate interaction.Site-directed mutagenesis indicated that six and ten residues were key for xylan and cellulose hydrolysis,respectively.Most of these crucial residues are centered on the subsites(-2 and-1)near the cleavage site,while residues playing moderately to slightly important roles were located at more distal regions of the binding cleft.Manipulating the latter residues could improve the activity of CbXyn10C on xylan and cellulose.The key residues for celluase activity are conserved across GH10 xylanases.Revisiting randomly selected GH10 enzymes revealed unreported cellulase activity,indicating that the dual function is a more common phenomenon than has been expected.It is noteworthy that the C-terminus of CbXyn10C/Ce148B,GH48,has both xylanase and cellulose activity.Does the synergy effect exist between C-terminus and N-terminus?In this study,we determined the hydrolyzing activities of GH10-CBM and CBM-GH48 on various substrates including cellulose,xylan,and corn straw.GH10-CBM and CBM-GH48 were found to degrade filter paper synergistically on the amount cellobiose and the intermolecular synergistic effect is 2.3.Intramolecular synergy effect was found on the degradation of xylan.The amounts for both of xylooligosaccharides and cellooligosaccharides hydrolyzed from corn straw were determined,and intermolecular synergy effect was observed,as reflected by the significantly elevated amount of cellobiose.Furthermore,immunofluorescence microscopy was used to explore whether GH10-CBM and GH48-CBM there is any synergistic effect on the untreated rice plant cell wall.These results provide innovation for the design of perfect and ideal CAZymes for degradation of cell wall polysaccharide.