Ursolic acid activates SIRT6 by enhancing enzyme-substrate interactions and promoting protein structural rearrangement.

Abstract

Ursolic acid (UA) has emerged as a promising bioactive compound with potential therapeutic effects, particularly in the upregulation of SIRT6, an important protein involved in various cellular processes, including longevity, stress response, and metabolism. Despite the growing interest in UA and its beneficial biological activities, the precise mechanisms governing its interaction with SIRT6 remain inadequately elucidated. This study aims to conduct a comprehensive investigation into the binding affinity of UA to SIRT6, as well as its effects on the protein's stability, kinetics, and structural characteristics. Molecular dynamics simulations using Schrodinger software analyzed parameters such as radius of gyration, RMSD, RMSF, and binding energy. The SIRT6 gene was cloned into the pET28a vector, expressed in Escherichia coli, and purified via affinity chromatography. Kinetic parameters (Km, Vmax, and Kcat) were assessed using fluorescence enzyme assays, while structural modifications were examined via fluorescence spectroscopy, FTIR, and UV-visible spectrophotometry. UA significantly enhances SIRT6 stability, reducing its radius of gyration and lowering binding energy from -25.38 to -47.93 kcal/mol. Kinetic analysis revealed a decrease in Km (13 to 10), an increase in Vmax (5013.42 to 9421.48 μM/min), and a rise in Kcat (15.03/s to 281.01/s), improving the Kcat/Km ratio. Structural assessments confirmed UA-induced modifications, increasing alpha-helix content (8.5 % to 26.2 %) and elevating the folding ratio from 0.066 to 14.8. However, it decreased aggregation index from 402.38 to 81.25. This integrative study elucidates UA's molecular influence on SIRT6, underscoring its potential therapeutic relevance across various signaling pathways.