# Molecular Dynamics Simulation Reveal the Structure-Activity Relationships of Kainoid Synthases
## 2 Results and Discussion
### Key Residues Responsible for Interacting with the Substrates  
At present, it has been widely recognized that key residues play an important role in the biosynthesis of terpenoids, such as aromatic residues stabilizing carbocation intermediates through \(\pi\)-cation interactions [11; 12]. In order to determine the key residues that kainoid synthases bind to on the substrates, we calculated the interaction energy between the substrate and each active site residue in 500 ns simulation, and analyzed the conformational changes of the key residues. We found that electrostatic interactions played a decisive role between kainoid synthases and substrates (Figure 12). In PnDabC, the Coulomb forces of 132R, 146K, 150R, 209K, 215R, 238T, and 363K and the Lennard-Jones potentials of 211F and 317F dominated binding to cNGG. In previous studies, GfKabC showed much more catalytic promiscuity, though the reason for this was not clear [8; 10]. Interestingly, we chose a 50 ns window in the catalytic process to analyze the conformational transformations of key residues, and found that after binding with cNGG, the benzene rings of 118F, 148F, 211F, 317F, and 359W rotated towards cNGG-1 COOH, and the rotation of the side chains of aromatic residues would not lead to any highly feasible substrate conformational change (Figure 13). This may be the reason for less catalytic promiscuity of PnDabC, and these conformational transformations improved the interaction with cNGG. In DsKabC, the benzene rings of 136F, 198F, and 302F rotated towards PKA-1 COOH, while in GfKabC, 107F, 124F, and 297F also rotated towards PKA-1 COOH. In the DsKabC-PKA complex, the aromatic amino acids were closer to PKA-1 COOH than in the GfKabC-PKA complex during the catalytic process. These findings suggest that residue-level conformational transitions of kainoid synthases also play a role in substrate binding and subsequent catalysis. From the analysis of the simulated endpoint kainoid synthase-substrate conformations, we found that three aromatic amino acids formed van der Waals forces with PKA in DsKabC, while the aromatic amino acids in GfKabC formed van der Waals forces and the hydrophobic interactions of \(\pi\)-cation and \(\pi\)-alkyl (Figure 14). According to the contribution analysis of residues, we found that the van der Waals forces of these residues were much stronger than the hydrophobic interactions (Table S1), so PKA in DsKabC was more stable during the catalytic process and DsKabC lacked catalytic promiscuity compared with GfKabC. We selected the conformational changes during the 250 ns to 300 ns period of the kainoid synthases catalytic process for further study (Figure 15). We found that, between 250 ns and 300 ns, PnDabC contained either more aromatic residues or more basic amino acids in its substrate-binding catalytic pocket compared to DsKabC and GfKabC. Aromatic residues can form \(\pi\)-cation interactions to stabilize substrates and intermediates, which may result in weaker catalytic promiscuity. In DsKabC, during the same period, the catalytic pocket contained either more aromatic residues or more basic amino acids than in GfKabC, making DsKabC more capable of forming \(\pi\)-cation interactions to stabilize substrates and intermediates. Conversely, the catalytic pocket of GfKabC contained the fewest aromatic residues and basic amino acids, lacking sufficient \(\pi\)-cation interactions to stabilize substrates and intermediates, making it less able to prevent alternative catalytic pathways and,  
Figure 12: Interaction energy between the substrate and kainoid synthases. thus, displaying stronger catalytic promiscuity. Additionally, at the midpoint of the simulation, GfKabC exhibited a more relaxed active pocket compared to PnDabC and DsKabC (Figure 16); here, we used the tool KVFinder for cavity detection and characterization of any type of biomolecular structure, with the results showing that GfKabC had a larger cavity volume than PnDabC and DsKabC (Table 1). The electrostatic potential energy of its active pocket was the lowest.