2.5.1 MD and conformational stability analysis
Based on the aforementioned experimental analyses of A355N, S356Y and D525N, MD simulations were performed on these three mutants to analyze their structural changes. The root mean squared deviation (RMSD) is the essential parameters to validate the quality of MD simulation. In the present study, the stability of wild type r-Rha1 and its mutants were determined by the deviations produced during the course of MD simulations. As illustrated in Fig. S5, r-Rha1 reached equilibrium after 4 ns of MD simulation, with a RMSD value of 0.5 Å, while A355N reached equilibrium after 11 ns, with a RMSD value of about 0.8 Å, and S356Y and D525N reached equilibrium after 5 ns, with a RMSD value of about 0.7 Å. The RMSD is used for measuring the differences between the backbones of a protein from its initial structural conformation to its final position.46 The higher RMSD values of three mutants than that of r-Rha1 suggested that mutants A355, S356Y and D525N can influence the backbone stability of the enzyme. A similar observation was reported earlier for several systems, including glycoside hydrolases and other enzymes. 49, 50