2.2 Molecular dynamic simulations
Through AMBER 18 program suite, NocTE-LSub and NocTE-DSub systems were subjected to molecular dynamic (MD) simulations using ff14SB force field23. Firstly, energy minimizations were carried out for these complexes to rationalize crystal structures. Afterwards, each system was heated from 0 K to 300 K and equilibrated for 50 ps under the isothermal−isobaric ensemble. Starting from this preprocessed conformation, six replicas of MD simulation trajectories were collected. Long-range electrostatic interactions and bond lengths in simulations were described using Particle Mesh Ewald (PME) method24 and the SHAKE algorithm25. Trajectory analyses, like root mean square deviation (RMSD) fluctuations and conformation distributions, were performed via the cpptraj module26 of AMBER 18.
For epimerization, the deprotonation distance d(H1901-LSub) was constrained between 3.00 Å and 3.50 Å using a harmonic vibrational potential with a force constant of 200 kcal/(mol·Å2) in the equilibration step for NocTE-LSub system, catching the critical pre-reaction structure for epimerization. Then the system was re-equilibrated without constraints and six replicas of 100 ns MD simulation trajectories were obtained. According to the distribution of the deprotonation distance and angle, two optimal trajectories md2 and md6 were selected and lengthened to 310 ns for the following mechanism investigations.