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

Glycogen branching enzymes (GBEs) catalyze branch formation in glucans, a process that requires precise recognition and positioning of donor and acceptor substrates; however, the structural basis of acceptor recognition in glycoside hydrolase family 13 subfamily 8 (GH13_8) GBEs remains unclear. In this study, a conserved groove characteristic of GH13_8 enzymes was identified, and alanine substitution in Clostridioides difficile 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.