Composite

Part:BBa_K5327037:Design

Designed by: Fangxian Chen   Group: iGEM24_BUCT   (2024-09-25)


MLS-CYP79F1(Truncated,Δter)-CYP83A1(Truncated)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2093
    Illegal SpeI site found at 2725
    Illegal SpeI site found at 3121
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2093
    Illegal SpeI site found at 2725
    Illegal SpeI site found at 3121
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2093
    Illegal BglII site found at 3761
    Illegal BamHI site found at 2851
    Illegal XhoI site found at 1497
    Illegal XhoI site found at 2446
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2093
    Illegal SpeI site found at 2725
    Illegal SpeI site found at 3121
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2093
    Illegal SpeI site found at 2725
    Illegal SpeI site found at 3121
    Illegal NgoMIV site found at 1189
    Illegal AgeI site found at 2329
    Illegal AgeI site found at 2593
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

To enhance the catalytic efficiency of the fusion protein involving CYP79F1 and CYP83A1, we integrated a linker between these two genes and added a mitochondrial targeting peptide (MLS) sequence at the N-terminus of the CYP79F1 gene. This modification allows the resulting fusion protein to be expressed and localized on the mitochondrial inner membrane, facilitating the catalysis of short-chain extended methionine dihomomethionine into the corresponding aldoxime (5-methylthiopentanal oxime) and contributing to the biosynthesis of both short-chain and long-chain aliphatic glucosinolates. Further oxidation of the aldoxime results in nitro acid compounds.


Rational Design and Evaluation Approach:

Building on the optimized enzyme subcellular localization strategy, we employed rational modifications to enhance the final product synthesis. The design involves:

1.Evaluation of Hydrophobicity and Hydrophilicity:We assessed the substrate channel's hydrophobic and hydrophilic properties in the wild-type and truncated protein structures and in the modified subcellularly localized fusion protein.
2.Molecular Docking and Dynamics Simulations:Using wild-type CYP79F1 and fusion proteins, we performed molecular docking and molecular dynamics simulations to identify key amino acid residues that interact with the substrates. We utilized GROMACS for the following:
Ligand and Receptor Preparation: Ligands were prepared using the GAFF (General AMBER Force Field), which ensures precise molecular interactions by categorizing atoms based on different chemical environments. The receptor was prepared using the AMBER99SB force field, which is widely used in protein structure simulations.
Simulation Environment Setup:A cubic closed simulation environment was established with a minimum distance of 1.0 nm between the molecule and the box edge. The SPC water model was used, and Na+ and Cl- ions were added to neutralize the system.
Energy Minimization: To optimize the system's geometry and reduce high-energy regions, energy minimization was performed using a combination of steepest descent and conjugate gradient methods.
Pre-Equilibration: NVT (constant volume and temperature) and NPT (constant pressure and temperature) equilibrations ensured the system's stability.
Molecular Dynamics Simulation: The final molecular dynamics simulation provided insights into the enzyme-substrate interaction sites and docking parameters.


Selective Amino Acid Modifications:

For CYP79F1 (trancated), we selected amino acids with high affinity for dihomomethionine and low affinity for aldoximes, prioritizing hydrophobic residues:

• Isoleucine (I): Highly hydrophobic and commonly interacts with other hydrophobic residues.
• Leucine (L): Highly hydrophobic, frequently found in hydrophobic core regions.
• Valine (V): Strong hydrophobicity, though slightly weaker than isoleucine and leucine.
• Phenylalanine (F): Strong hydrophobicity with an aromatic side chain that can engage in hydrophobic interactions.
• Tryptophan (W): Despite having a polar portion, it is overall highly hydrophobic, with an aromatic ring that can participate in hydrophobic interactions.

We prioritized leucine and isoleucine for modifications in CYP79F1 (trancated).

Fig 1. Rational modification sequence diagram of CYP79F1

Fig 2. The rational modification molecular docking comparison results display of CYP79F1

For CYP83A1 (trancated), we selected amino acids with high affinity for aldoximes and low affinity for dihomomethionine, focusing on polar residues:

• Glutamine (Q): Polar side chain (amide group), strong affinity for aldoximes.
• Asparagine (N): Polar side chain (amide group), high affinity for aldoximes.
• Serine (S) and Threonine (T): Polar side chains (hydroxyl groups), good affinity for aldoximes.
• Tyrosine (Y): Contains both polar hydroxyl and aromatic rings.

We chose glutamine and asparagine to modify CYP83A1 (trancated).

Fig 3. Rational modification sequence diagram of CYP83A1

Fig 4. The rational modification molecular docking comparison results display of CYP83A1

By comparing the mean and variance of RMSD values between two docking schemes, it was found that the parameters stabilized after the initial simulation phase, calculated from the final 200 ps. Analysis indicated a significant increase in the stability enhancement rate of enzyme-substrate docking reactions.

Fig 5. Computer-Assessed RMSD Data Representation of CYP79F1(trancated)

Fig 6. Computer-Assessed RMSD Data Representation of CYP83A1(trancated)
Table 1. Results of improved binding affinity and stability of P450 enzymes after rational modification

According to the results of molecular dynamics simulations, the binding stability of the two optimized P450 enzymes to the substrate increased by 31.22% and 29.46%, respectively. The results indicate that substituting the predicted binding site amino acids in CYP79F1 with leucine and in CYP83A1 with glutamine significantly enhances the catalytic efficiency and stability of the modified fusion protein.


Construction of Fusion Protein

To improve reaction efficiency, a linker peptide was inserted between the CYP79F1 and CYP83A1 genes, forming a more functional fusion protein. The introduction of the linker peptide optimizes protein interactions, enhancing enzyme reaction efficiency. The fusion protein facilitates more efficient substrate conversion, including the conversion of short-chain-elongated methionine to the corresponding aldoxime and involvement in aliphatic glucosinolate biosynthesis.

Fig 7. Rational modification sequence diagram of the fusion protein

Fig 8. Rational modification molecular docking comparison results display of fusion protein

Fig 9. SDS-PAGE image of the fusion protein


Modification of Subcellular Localization

To optimize enzyme subcellular localization, a mitochondrial targeting peptide (MLS) sequence was introduced at the N-terminus of the CYP79F1 gene, enabling the fusion protein to be localized and expressed on the mitochondrial inner membrane. This localization enhances protein expression efficiency in specific cellular regions and improves substrate conversion capability. Modifications targeting mitochondrial localization showed that the fusion protein more effectively catalyzed the conversion of dihomomethionine and further oxidized the resulting aldoxime to form an acid nitro compound. After construction, we incorporated the EGFP gene for mitochondrial laser confocal imaging, resulting in mitochondrial laser confocal images that confirm successful modification of subcellular localization.

Fig 10. The expression diagram of MLS-CYP79F1(Trancated,Δter)-CYP83A1(trancated)-EGFP

Fig 11. Mitochondrial laser confocal scanning images before and after rational modification

We can observe that under the guidance of the mitochondrial targeting sequence, the fusion protein successfully localized to the mitochondria.


Plasmid

Fig 12. The plasmid expression of MLS-CYP79F1(Trancated,Δter)-CYP83A1(trancated)


Source

Arabidopsis thaliana