# Rational Design and Modification of NphB for Cannabinoids Biosynthesis
## 2 Results
### Mechanistic Analysis of Production of Diverse Cannabinoids  
To elucidate the molecular basis for the enhanced catalysis of these mutants, we conducted a 100 ns molecular dynamics simulation for both the wild-type and the mutant, then carried out subsequent analysis after confirming the stability of the trajectory based on the root-mean-square deviation (RMSD) (Figure S4). we compared the wild-type NphB with the W3 variant, which showed significantly enhanced activity for four different aromatic substrates. As a promising aromatic prenyltransferase, NphB includes a cylindrical decameric \(\beta\)-barrel filled with solvent at its core, surrounded by \(\alpha\)-helices exposed to the solvent. The final helix at the C-terminus forms a lid over the barrel. The W3 variant contains six-point mutations distributed within the \(\beta\)-barrel and at its junction with the lid (Figure 4A). The Q161R mutation has led to an increase in hydrogen bonds with surrounding water molecules from three to seven, and the number of bound water molecules has increased from three in the wild-type to five. This enhancement significantly augments the hydrophobicity within the substrate pocket, thereby stabilizing the hydrophobic segment of the substrate (Figure 4B). The E214H mutation forms a hydrogen bond with the substrate's phenyl ring hydroxyl group, enhancing substrate stability. Additionally, a conformational change in the substrate occurs, reducing the distance between the GPP C1 and the olivetol C4 from 4.4 A to 3.5 A. This substantial reduction in the reactive distance between the two substrates promotes the occurrence of the reaction (Figure 4C). Mutation of residues 286 and 49 to cysteine facilitates the formation  
Figure 3: Selection of NphB mutants for the augmented biosynthesis of diverse cannabinoids. (**A**) CBG production by the wild-type and mutant strains. (**B**) CBGA production by the wild-type and mutant strains. (**C**) CBGV production by the wild-type and mutant strains. (**D**) CBGVA production by the wild-type and mutant strains. ND signifies not detected. The error bars represent standard deviation. Measurements were conducted in triplicate. Red represents the synthesis data of CBG, blue represents the synthesis data of CBGA, green represents the synthesis data of CBGV, and yellow represents the synthesis data of CBGVA. of a disulfide bond, which stabilizes the substrate-binding pocket and enhances the overall structural integrity of the enzyme. Moreover, the elevated local hydrophobicity resulting from this mutation is advantageous for the stabilization of the substrate conformation and the carbon cation intermediate (Figure 4D). Residues 47 and 290, situated at the entrance loop of the substrate-binding pocket, regulate substrate entry via conditional \(\alpha\)-helix dynamics. In the wild-type enzyme, H290 engages in hydrogen bonding with S46 and V48, thereby limiting the mobility of the \(\alpha\)-helix (Figure S5A). In the W3 mutant, the mutations H290D and V47D disrupt the pre-existing hydrogen bond network, leading to the formation of new interactions between D290 and I291 (Figure S5B). The reconfiguration of the hydrogen bond network diminishes the interactions of D290 with neighboring residues, alleviating the restraints imposed on the \(\alpha\)-helix. By analyzing the root mean square fluctuation (RMSF) values of WT and mutant W3, the RMSF value of residue F176 in the WT is 0.0376 nm, while in mutant W3 it is 0.044 nm, indicating that the residue has the least fluctuation and its side-chain spatial position is relatively stable (Figure S6). To quantify the motion of the \(\alpha\)-helix, we measured its distance to F176. The mutant exhibits an increased distance between the \(\alpha\)-helix and F176, which dilates the binding pocket and enhances the \(\alpha\)-helix flexibility, allowing for unimpeded substrate interaction (Figure 4E). This enhanced mobility facilitates greater contact between the substrate and GPP, thereby increasing the probability of the catalytic reaction.