AI-Assisted Identification of the Functional Residues of Ginsenoside Biosynthesis-Associated UGTs

Background/Objectives: Ginsenosides, one of the most pharmaceutically valuable chemical compounds in Panax ginseng, are synthesized with several enzymes, including UGTs. UGTs determine absorbability and physiological function upon consumption. Thus, understanding the functional residues of ginsenoside biosynthesis-associated UGTs is crucial for enhancing the production of valuable ginsenoside varieties. Methods: We collected the UGT homologs of high sequence similarity from two rate-limiting steps of the biosynthetic pathway. The 3D structures of these proteins were predicted using the AlphaFold3 model. The ligand-binding interactions of these UGTs were examined using SwissDock and CB-Dock2. Enzyme kinetics were analyzed with MPEK. Using these tools, we performed in silico mutagenic analyses to identify the functional residues of UGTs in detail. Results: We elucidated the molecular mechanisms of experimentally verified functional residues in UGTs, many of which were associated with optimal ligand interaction angles that expose target carbons. We also identified putatively important amino acid residues that mediate ligand interactions and modulate reaction kinetics by more than 25%. In this study, residues at positions 62, 224, 397, and 398 were shown to significantly influence enzyme kinetics. Conclusions: Our study provides the first structural analysis of the functional residues of ginsenoside biosynthetic UGTs based on their 3D structures. We identified several key amino acid residues essential for proper ginsenoside biosynthesis: (1) residues determining ligand interactions, (2) residues modulating ligand binding angles, and (3) residues affecting reaction kinetics. Our findings demonstrate an effective approach to identifying functional residues in plant enzymes and present valuable UGT candidates for future experimental validation.