2023_bit-china
In order to achieve high product yield, we considered modifying the biosynthetic enzymes for santalol and sclareol. We focused on three groups of compounds: FPP-STS, GGPP-TPS, copal-8-ol diphosphate-LPPS. For these three groups of enzymes and substrates that play a key role in the synthesis process, we identified the structure from the protein databank (PDB).
Molecular docking is a process of identifying the best matching pattern between two or more molecules through geometric matching and energy matching. Molecular docking has important application significance in enzyme engineering and drug design. We need to determine the binding free energy of molecular docking, the binding free energy is Gibbs free energy ΔG = -RTlnK, when the value of the binding free energy is negative, the system is stable. It can be understood that under isothermal isobaric conditions, after the receptor binds to the ligand, the system is more stable, and the lower the binding free energy, the more stable the system. We calculated the binding free energy data and analyzed their interactions using PyMol to identify key amino acid residues to guide site-directed mutation in subsequent experiments. The following picture shows the molecular interaction model between enzymes and their substrates.
The following table shows the binding free energy and Kd values of the three sets of substrates to the enzyme:
| Binding energy(kcal/mol) | Kd(uM) | |
|---|---|---|
| FPP-STS | -7.71 | 2.24 |
| GGPP-TPS | -6.57 | 15.3 |
| copal-8-ol diphosphate-LPPS | -8.32 | 0.791 |
According to the mutant binding free energy data, the mutation of Asp541 in the copal-8-ol diphosphate-LPPS to Arg reduces the binding free energy and increases the affinity. Other mutations cannot significantly increase affinity to the substrates. We found the Asp541Arg mutation in copal-8-ol diphosphate-LPPS are conducive to improving affinity, which provides guidance for subsequent enzyme engineering.
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