Part:BBa_K5327020

Dihomomethionine N-hydroxylase (Truncated)

Function:

The enzyme catalyzes the conversion of short-chain-extended methionine derivatives, specifically di-, tri-, and tetra-homomethionine, into their respective aldoximes—5-methylthiopentanal oxime, 6-methylthiohexanal oxime, and 7-methylheptanal oxime—within the inner mitochondrial membrane, thus laying the groundwork for subsequent metabolic engineering applications.

Usage and Biology

Genome localization:Chromosome: 1; NC_003070.9

Expression diagram:

Fig 1. The expression diagram of dihomomethionine N-hydroxylase (Truncated)

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of dihomomethionine N-hydroxylase (Truncated)

The PCR result:

Fig 3. The PCR result of dihomomethionine N-hydroxylase (Truncated)

Design Notes

Design Strategy: To optimize the functionality of the CYP79F1 gene, a strategy involving subcellular localization optimization was employed by removing the endoplasmic reticulum (ER) anchoring sequence. This modification enables the expressed protein to relocate from the ER to the cytoplasm, where it catalyzes the conversion of short-chain extended methionine derivatives—di-, tri-, and tetra-homomethionine—into their respective aldoximes: 5-methylthiopentanal oxime, 6-methylthiohexanal oxime, and 7-methylheptanal oxime. This modification lays the groundwork for subsequent applications in metabolic engineering. To enhance the catalytic efficiency of the modified enzyme and improve the synthesis of the final product, a semi-rational enzyme engineering approach was undertaken following the optimization of the enzyme’s subcellular localization. The modification strategy is outlined as follows: Initially, we assessed the hydrophobicity and hydrophilicity of the substrate channel in the wild-type CYP79F1 (short)(All mentions of "short" refer to "Truncated") enzyme and evaluated the truncated versions. The truncated proteins with modified sites were further assessed, followed by an evaluation of the fusion proteins with altered subcellular localization. Finally, selected modification sites were incorporated into the fusion proteins for comprehensive assessment. For the wild-type CYP79F1 (short) enzyme, molecular docking and molecular dynamics simulations were conducted to identify enzyme-substrate interaction sites, specifically the amino acid residues involved in binding. Molecular Simulation Workflow Using GROMACS:

1.Preparation of Ligand and Receptor:

Ligand Preparation: Ligand molecules were processed using the General AMBER Force Field (GAFF) for atomic type assignment. GAFF provides a detailed classification of atoms in diverse chemical environments, enhancing the force field’s accuracy in describing intra- and intermolecular interactions and ensuring precise receptor file preparation.

Receptor Preparation: The receptor protein was prepared using the AMBER99SB force field, widely recognized for its application in protein structure simulation, dynamics analysis, and free energy calculations. Notably, AMBER99SB excels in simulating protein folding and conformational dynamics and demonstrates excellent compatibility with the GAFF force field, significantly improving simulation efficiency.

2.Simulation Environment Setup:

Construction of the Simulation Box: A cubic, closed simulation box was constructed with a minimum distance of 1.0 nm between the solute and the box edge, positioning the receptor protein at the center.

Solvent and Ion Addition: The SPC (Simple Point Charge) water model, representing water as a rigid triangular structure, was employed, suitable for molecular dynamics simulations of biomolecules. To simulate physiological conditions, Na⁺ and Cl^- ions were added to neutralize the system’s total charge.

3.Energy Minimization:

Energy minimization was conducted to optimize the geometric structure and eliminate high-energy regions. A maximum of 10,000 iterations was set, with each step constrained to a maximum length of 0.01 nm and a convergence criterion of 1000.0 kJ/mol/nm. A combination of Steepest Descent and Conjugate Gradient methods was employed—first to rapidly reduce high-energy gradients, followed by more refined structural optimization.

4.Pre-equilibration Procedures:

NVT (constant number of particles, volume, and temperature) and NPT (constant number of particles, pressure, and temperature) equilibration phases were executed to ensure the system’s stability under controlled temperature and pressure, thus enhancing the accuracy of subsequent molecular dynamics simulations.

5. Molecular Dynamics Simulations:

After completing the preparation steps, molecular dynamics simulations were conducted to determine the enzyme-substrate interaction sites, along with detailed analysis of key parameters involved in the docking process.

Selective Design Strategy for CYP79F1 (short): For CYP79F1 (short), amino acids with low affinity for aldoximes and high affinity for dihomomethionine were selected for site-specific modification. The prioritized amino acids are as follows:

• 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.

Prioritization Ranking:

1.Isoleucine (I)

2.Leucine (L)

3.Valine (V)

4.Phenylalanine (F)

5.Tryptophan (W)

Fig 4. Rational modification sequence diagram of CYP79F1

Fig 5. The rational modification molecular docking comparison results display of CYP79F1

Subsequently, isoleucine (I) and leucine (L) were selected for semi-rational modification of CYP79F1 (short). The modification results are as follows: Through predictive data analysis and affinity scoring functions within AutoDock, it was found that substituting the predicted binding sites with leucine (L) yielded the best modification results. Molecular docking results revealed that the modified enzyme, with leucine replacing the original binding sites, exhibited a tighter and more stable interaction with dihomomethionine due to the enhanced hydrophobicity of the binding sites. The modified enzyme displayed increased affinity for dihomomethionine while showing reduced affinity for aldoximes, thereby improving the reaction's stability. The modified enzyme also formed hydrogen bonds with the substrate, effectively enhancing polar interactions between the enzyme and substrate, increasing the binding surface area, and subsequently boosting the reaction rate. This modified enzyme model was experimentally validated to assess its impact on sulforaphane production in Saccharomyces cerevisiae, with observed improvements in the final product yield.

Plasmid

Fig 6. The plasmid expression of dihomomethionine N-hydroxylase (Truncated)

Source

Arabidopsis thaliana

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1085
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1085
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1085
  Illegal XhoI site found at 489
  Illegal XhoI site found at 1438
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1085
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1085
  Illegal NgoMIV site found at 181
  Illegal AgeI site found at 1321
  
• 1000
  COMPATIBLE WITH RFC[1000]