A Push-Pull Loop Motif Controls Product Distribution in GH5 Endocellulases

Endocellulases are central to cellulose deconstruction but are commonly viewed as stochastic catalysts whose product distributions are difficult to control. Here, we report a loop encoded mechanism that governs cleavage-site selection in GH5 endocellulases. By comparing two closely related enzymes with distinct product profiles, we show that a short loop near the binding cleft determines substrate positioning and hydrolytic outcomes. Molecular dynamics simulations reveal that a triaspartate loop (DDD) promotes deep substrate binding and favors central bond cleavage, whereas an alternative loop (DND) conformation restricts substrate entry and leads to nonspecific hydrolysis. Minimal loop substitutions are sufficient to reversibly switch product distributions without altering the catalytic core, and the same behavior is retained on regenerated amorphous cellulose. These results demonstrate that endocellulase product profiles can be rationally programmed through loop engineering and functionally coupled to phosphorylated sugar biosynthesis requiring controlled oligosaccharide inputs.