Engineering galactoside acetyltransferase for enhanced hesperetin-7-O-glucoside bioavailability

Flavonoid glycosides exhibit compromised bioavailability due to low membrane permeability. To address this limitation, we acetylated flavonoids through enzymatic reactions to increase bioavailability. This study first reported that Hesperetin-7-O-glucoside (Hes-7-G) was acetylated by galactoside acetyltransferase (GAT), and the apparent permeability (P_app) of the Caco-2 monolayer was increased by 69%, indicating the acetylated Hes-7-G application potential to improve bioavailability. Subsequently, we designed GAT mutants through comprehensive computational and experimental methods to improve the acetylation efficiency and elucidate the catalytic mechanism. Molecular Dynamics (MD) simulations found that Tyr483 and Met127 are key residues that control flavonoid binding through dynamic van der Waals interactions, while His115 and Thr113 mediated proton transfer accounts for 85-90% of the catalytic activity. Rational substitution of Pro148 with alanine (P148A) increased the flexibility of the cofactor binding ring and increased the catalytic efficiency (K_cat/K_M) by 21%. Average non-covalent interaction (aNCI) analysis revealed that regional selectivity in the glucose portion was controlled by hydrophobic interactions with Tyr483 and hydrogen bonding with Gly125, and rhamnose substitution caused spatial conflict. This work deciphered the structure-activity relationship of GAT, established a framework for protein engineering, and highlighted enzyme-driven acetylation as a sustainable strategy for optimizing flavonoid pharmacokinetics. KEY POINTS: • Engineered acetyltransferase enhances flavonoid glycoside absorption. • P148A mutation improves catalytic efficiency. • Insight into the catalytic mechanism of GAT by flavonoid glycoside substrates.