AiCE-Guided Loop Dynamics Redesign in a Novel Glycosyltransferase Enhances the Dual-Regioselective Glycosylation of Rhein

Abstract Cassia seeds contain bioactive anthraquinones, including rhein and its glycosides, but their efficient production is limited by the low activity and poor regioselectivity of natural enzymes. Here, we identified NtUGT75C1 from Nicotiana tomentosiformis as a glycosyltransferase producing rhein-8-O-β-d-glucoside and rhein-1-O-β-d-glucoside. Loop dynamics were redesigned using the AI-informed constraints for protein engineering (AiCE) framework. The S55P mutant increased 8-O-glycoside formation more than 2-fold and also increased 1-O-glycoside formation. It displayed more than 80% of its maximum activity when assayed at 50 °C, and its nominal kcat/Km was 2.54-fold that of WT. Molecular dynamics simulations indicated increased local rigidity and redistributed flexibility, consistent with possible long-range allosteric coupling and a more localized low-free-energy basin. These findings identify an engineered glycosyltransferase candidate for modifying functional food components and demonstrate the utility of AiCE-guided loop engineering.