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

Area of Science:

• Biochemistry
• Pharmacology
• Enzyme Engineering

Background:

• Flavonoid glycosides possess poor bioavailability due to limited membrane permeability.
• Enzymatic acetylation offers a strategy to enhance the absorption of these compounds.

Purpose of the Study:

• To investigate the enzymatic acetylation of Hesperetin-7-O-glucoside (Hes-7-G) using galactoside acetyltransferase (GAT).
• To engineer GAT mutants for improved acetylation efficiency and understand its catalytic mechanism.
• To enhance flavonoid glycoside bioavailability through enzyme-driven modification.

Main Methods:

• Enzymatic acetylation of Hes-7-G using GAT.
• Caco-2 cell monolayer assays to measure apparent permeability (P_app).
• Computational methods including Molecular Dynamics (MD) simulations and Average non-covalent interaction (aNCI) analysis.
• Site-directed mutagenesis (P148A) to engineer GAT.

Main Results:

• Acetylated Hes-7-G showed a 69% increase in P_app across Caco-2 cell monolayers.
• MD simulations identified key residues (Tyr483, Met127, His115, Thr113) involved in substrate binding and catalysis.
• The P148A GAT mutant exhibited a 21% increase in catalytic efficiency (K_cat/K_M).

Conclusions:

• Enzyme-driven acetylation is a viable strategy for improving flavonoid glycoside bioavailability.
• Understanding the structure-activity relationship of GAT enables protein engineering for enhanced enzymatic function.
• This study provides a framework for optimizing pharmacokinetic properties of natural compounds via protein engineering.