# Molecular Dynamics Simulation Reveal the Structure-Activity Relationships of Kainoid Synthases
## 1 Introduction  
The kainoids (1) are 2,3,4-trisubstituted pyrrolidine derivatives that contain a glutamic acid moiety and various substituents at the four-position of the pyrrolidine ring (Figure 1). Domic acid (DA) (3) [1] and kainic acid (KA) (2) [2], the representatives of the kainoids, were first isolated from Japanese marine algae _Chondria armata_ and _Digenea simplex_. Both types of seaweed are used as insect repellents in traditional Chinese and Japanese medicine. Later, it was found that DA can also be synthesized in some diatoms, such as _Pseudonitrilexchia_ and _Nitzschia_[3], while KA can be synthesized in some red algae, like _Grateloupiq filicina_. DA and KA act as excitatory amino acid receptor agonists, and KA is widely used as a tool in neuropharmacology to stimulate nerve cells and mimic disease states, such as epilepsy [4], Alzheimer's disease, and Huntington's chorea [5]. As members of a novel class of marine meroterpenoids, the basic medical research application and biosynthetic pathway of DA and KA have attracted considerable attention in recent years [6; 7; 8].  
DA and KA are currently synthesized by chemical or biological techniques. KA can be successfully synthesized by constructing 2,3,4-tri-substituted pyrrolidine scaffolds through SMI2-promoted stereocomplementary ring closure reactions [9]. The key structure of the C1\({}^{\prime}\)-C2\({}^{\prime}\) Z-configuration of DA is completed using the unsaturated lactam structure.  
Three continuous stereocenters on the pyrrolidine ring are effectively established through a sequential rearrangement reaction, and side chain fragments are introduced by modifying the Julia-Kocienski reaction [10]. Compared with chemical synthesis, biosynthesis frequently shows higher regional and stereoselectivity. In the DA biosynthetic pathway, isopentenyltransferase (IPT) uses geranyl pyrophosphate (GPP) and L-Glu as substrates to synthesize L-NGG. Then, 7\({}^{\prime}\)-carboxy-L-NGG (cNGG) is synthesized by the cytochrome P450 enzyme. Finally, isodomic acid is synthesized by kainoid synthases PnDabC and then converted into domoic acid [6; 7]. In the KA biosynthetic pathway, IPT uses dimethylallyl pyrophosphate (DMAPP) and L-Glu as substrates to synthesize prekainic acid (PKA). Then, KA is synthesized by kainoid synthases DsKabC and GfKabC. Kainoid synthases have been found to have catalytic promiscuity [8], among which, GfKabC has been found to have higher catalytic promiscuity. Understanding the relationship between the structure, dynamics, and function of kainoid synthases is essential for the rational design and effective control of DA and KA biosynthesis.  
In this study, we used structural prediction, molecular docking, and molecular dynamics simulations (MDS) to investigate the conformational dynamics of kainoid synthases. Firstly, AlphaFold2 was used to predict the tertiary structures of three kainoid synthases: PnDabC, DsKabC, and GfKabC. Molecular docking was then performed using AutoDock. Secondly, the MDS over 500 ns showed that there were binding and catalytic processes in the entire simulation. The conformational transformations of key residues of kainoid synthases played important roles in the binding and catalytic processes. Finally, we found that the relaxation degree of the active pocket, the number and rotation of aromatic residues interacting with substrates during catalysis, the number of potential \(\pi\)-cation bonds, and the differences in binding energy may contribute to the promiscuity of kainoid synthases.