The N-Terminal α-Helical Domain of GH13_8 Glycogen Branching Enzymes Influences Short-Chain Substrate Utilization and Short-Branch Formation.

Glycogen branching enzymes (GBEs) catalyze the formation of α-1,6 glycosidic branches in glycogen, thereby influencing glycogen structure and physicochemical properties. Some bacterial GH13_8 GBEs can act on short maltooligosaccharides, unlike GH13_9 GBEs, which act on long substrates. The major structural difference between the two subfamilies lies in the N-terminal α-helical region of GH13_8 enzymes, positioned near the substrate entrance. The GH13_8 GBE from Clostridioides difficile was selected for structure-function analysis of its N-terminal α-helices. Mutational and truncation analyses demonstrated that these helices influence substrate accessibility and short-branch formation. Molecular dynamics simulations identified residues in the N-terminal α-helical region likely involved in substrate interactions. Guided by these findings, two variants, I12S and M27S, were engineered, producing more short-branched products and exhibiting 153% and 132% of wild-type activity, respectively. These findings identify the N-terminal α-helices as a promising engineering target for promoting short branch formation and generating denser α-glucan architectures.