Key Loops in GH13_8 Subfamily Glycogen Branching Enzymes Modulate Their Branch Length Preference

Glycogen branching enzymes (GBEs) catalyze the formation of α-1,6-glycosidic linkages, introducing branch points into α-glucans such as glycogen and starch. Branch length directly affects the structural, functional, and nutritional properties of these polysaccharides, yet the structural determinants underlying GBE specificity remain incompletely understood. In this study, we investigated the functional roles of three conserved loops─near residues 104, 185, and 454─positioned close to the branch-chain binding groove in the glycoside hydrolase (GH) family 13_8 and GH13_9 GBEs from Anaerococcus prevotii (Ap). Sequence and structural analyses revealed that loop 185 is highly conserved across both subfamilies, while loops 104 and 454 are significantly longer in GH13_8 GBEs, with loop 454 showing the most pronounced difference. To probe their influence on branch length specificity, we engineered ApGBE13_9 by replacing its native loops 104 and 454 with their longer counterparts from ApGBE13_8. Substituting loop 104 alone had no effect on long-branch formation (degree of polymerization (DP) 6–8), while loop 454 replacement shifted activity toward short-branch production (DP 3–4). Dual substitution broadened the branch length range (DP of 3–8), indicating a synergistic interaction between the two loops. Molecular dynamics simulations revealed that the increased flexibility of the longer loop 454 (from ApGBE13_8) perturbs loop 185, causing it to form a “blocked-like” conformation that hinders branch-chain extension within the branch-chain binding groove and promotes the formation of short branches. Long loop 104 acts cooperatively with loop 454 (both from ApGBE13_8), functioning as a dynamic “door” that modulates access to the branch-chain binding groove and enables dual branch length production. These findings provide mechanistic insight into GBE specificity and support rational enzyme engineering for tailored α-glucan synthesis.