His70 of Acetivibrio alkalicellulosi Cel5A is important for efficient hydrolysis of short cellodextrins.

Cellulose, a linear glucan linked by β-1,4 glycosidic bonds, is the most abundant renewable polysaccharide on earth. Complete enzymatic hydrolysis of cellulose liberates the readily metabolizable glucose that could be further converted to valuable biocommodities, and essential to this process are cellulases that hydrolyze the β-1,4 glycosidic bonds. Cellulases are among the most intensively studied and best understood enzymes, and many key residues have been uncovered and interrogated with respect to their functions in catalysis and/or substrate binding. However, it remains to be explored whether additional residues, especially in many poorly characterized cellulases such as processive endoglucanases, might also be functionally important. Here, we investigated a processive endoglucanase from an alkaliphilic bacterium Acetivibrio alkalicellulosi AaCel5A that consists of a glycohydrolase family 5 (GH5) domain and two tandem carbohydrate-binding module family 6 (CBM6) domains. Via structure-guided engineering, we uncovered the functional importance of a previously underexplored but relatively conserved histidine (histidine70 or His⁷⁰). His⁷⁰ itself appears to be largely dispensable for hydrolyzing β-1,4 glycosidic bonds, but it is important for efficient hydrolysis of short cellodextrins such as cellotriose, cellotetraose, and cellopentaose, likely through its ability to coordinate substrate binding. Our work thus provides important mechanistic insights into how processive endoglucanases may act on short cellodextrins.