All data support published article "Function of four tryptophan residues on Cel7A catalytic efficiency: insight into cellulase screening strategy based on natural cellulose or cellulose analogs"

There is a high level of conservation of tryptophans within the active site architecture of the cellulase family, whereas the function of the four tryptophans in the catalytic domain of Cel7A is unclear. By mutating four tryptophan residues in the catalytic domain of Cel7A from Penicillium piceum (PpCel7A), the binding affinity between PpCel7A and p-nitrophenol-D-cellobioside (pNPC) was reduced as determined by Michaelis–Menten constants, molecular dynamics simulations, and fluorescence spectroscopy. Furthermore, PpCel7A variants showed a reduced level of cellobiohydrolase activity against cellulose analogs or natural cellulose. Therefore, it could be concluded four tryptophan residues in Cel7A played a critical role in substrate binding. Mutagenesis results indicated that the W390 stacking interactions at the -2 site played an essential role in facilitating substrate distortion to the -1 site. As soon as the function was altered, the mutation would inevitably affect the catalytic activity against the natural substrate. Interestingly, no clear relationship was found between the cellobiohydrolase activity of PpCel7A variants against pNPC and Avicel. pNP contains many electrophilic groups that may result in overestimation of the binding constant between tryptophan residues and pNPC in comparison to the natural substrate. Consequently, screening improved cellulase using cellulose analogs would divert attention from the target direction for lignocellulose biorefinery. Clarifying mechanism of catalytic diversity on the natural cellulose or cellulose analogs may give better insight into cellulase screening and selecting strategy.