Abstract
Bifunctional glycoside hydrolases have potential for cost-savings 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 cello-oligosaccharides. 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 structures with xylo-oligosaccharides, seven sugar-binding subsites were found, and many residues responsible for substrate interactions were identified. Site-directed mutagenesis indicated that 6 and 10 amino acid residues were key residues for xylan and cellulose hydrolysis, respectively. The most important residues are centered on subsites -2 and -1 near the cleavage site, whereas residues playing moderate roles could be located at more distal regions of the binding cleft. Manipulating the residues interacting with substrates in the distal regions directly or indirectly 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.
Highlights
Bifunctional glycoside hydrolases have potential for cost-savings in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals
Glucose can be readily fermented into ethanol, and the five-carbon xylose is a feedstock of biofuels and bio-based chemicals, thanks to the vast progress achieved in recent years in engineered yeasts, Escherichia coli, and other alternative microbes [1, 2]
Isothermal titration calorimetry (ITC)5 can be used to characterize the number of substrate-binding subsites [18]
Summary
Isothermal titration calorimetry (ITC) can be used to characterize the number of substrate-binding subsites [18]. The binding constants (Ka) of E140Q/E248Q to xylotriose to xylopentaose were 500 –700 ϫ 103 MϪ1, ϳ10-fold higher than that for xylobiose but ϳ2-fold lower than that for xylohexaose This suggests that CbXyn10C has at least six sugar-binding subsites for xyloseconfigured substrates. The catalytic efficiency of CbXyn10C with xylo- and cello-oligosaccharides was determined. Low catalytic efficiency was observed for cellopentaose (0.009 mMϪ1 sϪ1), which increased by 4.4-fold for cellohexaose (Table 2) Both the binding constants (as determined in ITC) and catalytic efficiencies increased with higher degrees of polymerization for either configuration of the oligosaccharides, we noticed that the catalytic efficiencies from X3 to X6 steadily increased, whereas the binding constants for X3 to X5 appeared to be at the same magnitude.
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