# Molecular Dynamics Simulation Reveal the Structure-Activity Relationships of Kainoid Synthases
## 2 Results and Discussion
### Overall Stability of the Kainoid Synthes-Substrates Complex  
Root mean square deviation (RMSD) represents the sum of atomic deviations between all conformations and target conformations at a certain time. It is an important basis for measuring system stability. In the substrate-bound systems, the RMSD values of PnDabC and DsKabC converged at around 0.40 nm and 0.53 nm, respectively, indicating that the complex structures were gradually stabilizing (Figure 4). For the first 350 ns, the RMSD value of the GfkabC-PKA complex rose rapidly and converged to 0.45 nm, and finally experienced a large fluctuation during 350-500 ns. The RMSD of GfKabC was not synchronized with the complex after 350 ns, indicating that the RMSD fluctuation of the complex came from the conformational change of PKA binding to GfKabC. The trajectory of PKA binding to GfKabC in the complex was analyzed (Figure 5). At the time points 0, 350, 400, and 500 ns, the distance between PKA and the Fe atom increased from the initial 8.8 nm to the final 32.8 nm. This indicates that PKA gradually separated from the initial binding site after 350 ns. Figure 4: RMSD of kainoid synthases–substrates, kainoid synthases, and substrates. Figure 5: The conformational change of GfKabC-PKA (the purple stick–ball model is PKA, the black ball model is Fe\({}^{2+}\), the purple dash is the interaction between the GfKabC and PKA, GfKabC coloring style for rainbow). In order to analyze the state of the substrate on the surface of the kainoid synthase and obtain the initial docking site of the substrate, we analyzed the distance between the center of mass (Fe\({}^{2+}\)) of the initial docking site and the center of the substrate, as well as the distance between the substrate and the center of mass of the kainoid synthase. These distances reflect the binding state of the substrate to the kainoid synthase. The distance between cNGG and PnDabC, and the distance between cNGG and Fe\({}^{2+}\), gradually stabilized, indicating that the binding between cNGG and PnDabC was gradually stabilizing (Figure 6). Although the distance between PKA and Fe\({}^{2+}\) fluctuated within the first 100 ns, the fluctuation value did not exceed 1 nm. The distance between the PKA and DsKabC center and Fe\({}^{2+}\) gradually stabilized, indicating that the combination of PKA and DsKabC was gradually stabilizing. However, the distance between PKA and GKabC, and the distance between PKA and Fe\({}^{2+}\), remained stable within the first 350 ns, indicating that the binding of PKA and GfKabC was relatively stable during this period. After 350 ns, these distance values gradually showed relatively drastic fluctuations, indicating that the binding between PKA and GfKabC was no longer stable. The radius of gyration (Rg) can be used to characterize the tightness of the complex structure. According to the analysis, the Rg of the PnDabC-cNGG complex rose slowly and then remained stable, indicating that the overall structure of the complex was gradually stabilizing. The Rg fluctuation amplitude of the DsKabC-PKA complex decreased and stabilized gradually. For the GfKabC-PKA complex, the Rg gradually decreased over the first 350 ns, then suddenly increased and experienced sharp fluctuations after 350 ns, indicating that the overall structure of the GfKabC-PKA complex remained stable in the 0-350 ns range (Figure 7). In the range of 350-500 ns, the overall Rg increased and fluctuated as PKA departed from the initial binding site of GfKabC. Superposition of the substrate with kainoid synthases revealed that cNGG in PnDabC and PKA in DsKabC had a high degree of overlap, meaning the substrate was distributed at the initial binding site, indicating the binding of cNGG with PnDabC and PKA with DsKabC was stable. In GfKabC, PKA was distributed in other locations on the surface of GfKabC, indicating that there were both stable and unstable periods in the combination of PKA and GfKabC (Figure 8). Figure 6: Distance between the substrate and kainoid synthase.