Bipartite Anchoring at the +3 Subsite Governs Trisaccharide Specificity in a PL7 Alginate Lyase and Is Transferable to a Homologous Enzyme

Alginate lyases produce alginate oligosaccharides (AOS) whose bioactivities depend on the degree of polymerization (DP). Loop regions and individual residues influence product DP. However, how residues from loops cooperate at subsites to enforce a defined DP remains unresolved. Here, we identified ALG4, a PL7_5 alginate lyase specifically producing trisaccharides (ΔDP3), and ALG3, a same-subfamily homologue generating ΔDP2−ΔDP4 mixtures. Action-pattern analysis showed that ALG4 accommodates three sugar residues at the +1/+2/+3 subsites without extension toward +4. Structurally, the Orange Loop and Red Loop converge beyond +3 to form a distal occlusion zone in ALG4, absent in ALG3. Molecular dynamics (MD) simulations and systematic experiments supported a bipartite anchoring mechanism comprising a physical barrier layer (Y112/E56) and a direct binding layer (R105/K38) coupled through a hub network. Gain-of-function transplantation of these elements into ALG3 progressively shifted products toward trisaccharides, demonstrating a transferable residue-level design strategy within this homologous enzyme pair.