Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase

[Display omitted] •Structural mechanism of cellulose depolymerization by GH48 cellulase was examined.•BlCel48B has high processivity against crystalline and amorphous cellulose.•Substrate size and surface morphology dictate the hydrolytic rate, enzyme binding, and processivity.•Clusters of co-evolved residues drive the recognition and strength of cellulose binding to the active site.•Enzyme low-turnover and high processivity rates correlate with the rigidity of the catalytic tunnel. Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.