Background: Aromatic residues line glycoside hydrolase active sites mediating ligand binding. Results: Binding affinity is significantly altered upon tryptophan to alanine mutation, although relative to the location in the active site. Conclusion: Aromatic-carbohydrate interactions are employed in a variety of functionalities within the purview of ligand binding. Significance: Understanding the functional role of aromatic residues in the active site is necessary for the rational design of new carbohydrate-active enzymes.

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study.

In nature, processive and non-processive cellulase enzymes deconstruct cellulose to soluble sugars. From structural studies, the consensus is that processive cellulases exhibit tunnels lined with aromatic and polar residues, whereas non-processive cellulases exhibit open clefts with fewer ligand contacts. To gain additional insight into the differences between processive and non-processive cellulases, we examine the glycoside hydrolase family 7 (GH7) cellobiohydrolase, Cel7A, and the endoglucanase, Cel7B, from Trichoderma reesei with molecular simulation. We compare properties related to processivity and compute the binding affinity changes for mutation of four aromatic residues lining the Cel7A active site tunnel and Cel7B cleft to alanine. For the wild-type enzymes, dissimilar behavior is observed at nearly every glucopyranose-binding site from -7 to +2, except in the -2 site, suggesting that the structural differences directly around the catalytic center and at the active site tunnel entrances and exits may all contribute to processivity in GH7s. Interestingly, the -2 site is similar in both enzymes, likely due to the significant conformational change needed in the cellodextrin ligand near this site for catalysis. Moreover, aromatic residue mutations in the Cel7A and Cel7B active sites display only small differences in binding affinity, but the ligand flexibility and enzyme-ligand interactions are only locally affected in Cel7A, whereas the entire ligand is significantly affected when any aromatic residue is mutated in Cel7B.

Background: Family 1 carbohydrate-binding modules (CBMs) are often components of cellulases for binding to cellulose. Results: Family 1 CBM binding affinity is dramatically affected by the presence of O-glycosylation near the CBM binding face. Conclusion: Glycosylation should be accounted for in CBM binding affinity studies. Significance: Glycosylation can be harnessed to tune cellulase binding affinity, which is known to affect activity.