Identification of Key Residues Governing Branch Activity and Acceptor Substrate Binding in GH13_8 Glycogen Branching Enzymes

GBE13_8 revealed five residues that are critical determinants of branching activity. These results confirm the groove as the acceptor-binding site and support a "Y"-shaped substrate routing model distinct from the "h"-shaped model in GH13_9 GBEs. Structure-guided analysis further enabled engineering of this region, yielding a W473H variant with a 24% increase in catalytic activity. These findings define the structural determinants of acceptor recognition in GH13_8 GBEs and provide structural insights that may facilitate future engineering of GH13_8 enzymes for applications in starch modification.