# Molecular Dynamics Simulation Reveal the Structure-Activity Relationships of Kainoid Synthases
## 2 Results and Discussion
### Key Structural Moieties of the Substrate Recognized by Kainoid Synthases  
In order to determine the key structural part of the substrates recognized by kainoid synthases, we counted the number and frequency of hydrogen bonds between the individual functional groups of kainoid synthases and substrates in 500 ns (Figure 11). For PnDabC, we found three carboxyl groups of cNGG, namely cNGG-1 COOH, cNGG-2 COOH, and cNGG-3 COOH, were always the key sites for recognition, the cNGG-3 COOH from the geranyl pyrophosphate formed the most hydrogen bonds with the PnDabC. In total, 29 hydrogen bonds were formed between PnDabC and cNGG; among them, 12 hydrogen bonds were accepted by the cNGG-3 COOH from geranyl pyrophosphate, for which the donors were 119Q, 150R, 209K and 363K, respectively. The two carboxyl groups, cNGG-1 COOH and cNGG-2 COOH, from L-Glu accepted 22 hydrogen bonds donated by 146K, 215R, 371Y, 132R, 238T, 239S, and 235H, respectively. Among them, cNGG-1 COOH accepted seven hydrogen bonds donated by 146K, 215R, and 371Y, while cNGG-2 COOH accepted 10 hydrogen bonds donated by 132R, 238T, 239S, and 235H. According to the frequency of hydrogen bond formation, it can be divided into two periods; in the first 200 ns, cNGG mainly accepted hydrogen bonds donated by 371Y, 235H, 238T, 239S, 150R, and 209K, indicating that it may be the binding stage of cNGG and PnDabC. The higher the occupancy, the more critical residues needed to bind. In the latter 300 ns, cNGG mainly formed hydrogen bonds with 119Q, 150R, 132R, 146K, 215R, 239S, and 363K, among which, the occupancy of cNGG-3 COOH and 363K reached 59.3%, revealing that 363K is the key residue in the catalytic process of PnDabC. Compared with PnDabC, DsKabC formed fewer hydrogen bonds with PKA. PKA has two carboxyl groups, and only PKA-1 COOH accepted hydrogen bonds donated by 134K and 202R.  
Furthermore, the nitrogen atom of PKA donated hydrogen bonds to 304E. After 140 ns, the PKA-1 COOH accepted stable hydrogen bonds donated by NH1 and NH2 of 202R, and the average occupancy rates were 43.5% and 68%, respectively. The OE1 of 304E also accepted two stable hydrogen bonds donated by nitrogen atoms of PKA. Compared with PnDabC and DsKabC, the hydrogen bond between GfKabC and PKA only existed in the first 400 ns, and only PKA-1 COOH in two carboxyl groups accepted hydrogen bonds donated by 3 arginine. Furthermore, nitrogen atoms of PKA donated hydrogen bonds to 299E and 302D, respectively. In the first 200 ns, 122R and 196R donated hydrogen bonds to PKA-1 COOH, respectively. The highest hydrogen bonds occupancy was 42.8%, which was lower than PnDabC and DsKabC. After 200 ns, the hydrogen bonds between PKA and GfKabC were volatile, and there were no hydrogen bonds after 400 ns. The hydrogen bond occupancy and hydrogen bond receptors of DsKabC and GfKabC showed that \(\gamma\)-carboxyl group of PKA was the key recognized group. For PnDabC, the three carboxyl groups of cNGG were all key recognized groups.  
Figure 10: Buried SASA of kainoid synthases.  
Figure 11: Hydrogen bond occupancy between substrates and kainoid synthases.