Part:BBa_K4625911

MHETaseS491A

The MHETase gene is a gene found in the bacterium Ideonella sakaiensis, which is involved in the degradation and assimilation of the plastic poly(ethylene terephthalate) (PET). The gene encodes for an enzyme that catalyzes the hydrolysis of MHET to produce terephthalic acid (TPA) and ethylene glycol (EG). We have incorporated S491A mutation in this gene which proved to be better than the WT in in-silico experiments. The parameters that were used to assess the stability and efficiency of the mutation are docking scores, RMSD and RMSF values from Molecular Dynamics Simulations, and free energy calculations using MM/PBSA. Interested researchers can use it in their projects by different kinds of expression and expression studies with different chassis. This sequence is codon optimised for expression in E.coli.

Usage and Biology

The reaction mechanism of MHETase has been studied in several research articles. The mechanism starts with MHET binding to the active site of MHETase, forming an acyl-enzyme intermediate. The conversion of MHET occurs in two steps, with a rate-limiting step activation barrier of Delta G double dagger = 19.35 +/- 0.15 kcal[1]. The MHETase structure suggests a serine hydrolase mechanism for MHET hydrolysis. In deacylation, His528 plays a role in restoring the catalytic serine, transferring a proton from a water molecule to Ser225 and generating a free. The positioning of the substrate in the active site of MHETase is reminiscent of the tannin acyl α/β-hydrolase from L. plantarum bound to ethyl gallate. The enzyme is applicable over a broad pH range, with high activities from pH 6.0 to 9.5, and the activity increases with rising temperature up to 44 °C after which the enzyme is rapidly inactivated.[2]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal BamHI site found at 559
  Illegal XhoI site found at 1817
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1
  Illegal NgoMIV site found at 244
  Illegal NgoMIV site found at 358
  Illegal NgoMIV site found at 814
  Illegal AgeI site found at 311
  Illegal AgeI site found at 428
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental Validation

This part is validated through three kinds of in silico analysis: Molecular Docking, Molecular Dynamics Simulation and MM/PBSA.

Molecular Docking

Docking was performed using AutoDock Vina[3,4].

Molecular Dynamics Simulation

Simulations were performed by GROMACS 2023 [5] using CHARMM27 forcefield and TIP3P water model for 100ns at 303.15K and a pressure of 1 atm. The solvated protein ligand system consisted of 63876 atoms.

MMPBSA

In order to calculate free energy of protein-ligand system, gmx_MMPBSA package was used incorporating the GB protocol.[6,7]

Results

Docking Scores

MD Simulations

1. RMSD (backbone)

2. RMSD (ligand with respect to backbone)

3. RMSF

MMPBSA

WT: -19.53 kcal/mol

S491A: -21.60 kcal/mol

References

1. Alexandre V. Pinto, Pedro Ferreira, Rui P. P. Neves et al. Reaction Mechanism of MHETase, a PET Degrading Enzyme. ACS Catalysis 2021 11 (16), 10416-10428. DOI: 10.1021/acscatal.1c02444

2. Brandon C. Knott, Erika Erickson, Mark D. Allen, +16, and John E. McGeehan. Characterization and engineering of a two-enzyme system for plastics depolymerization. 117 (41) 25476-25485. https://doi.org/10.1073/pnas.2006753117

3. Eberhardt, J., Santos-Martins, D., Tillack, A.F., Forli, S. (2021). AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. Journal of Chemical Information and Modeling.

4. Trott, O., & Olson, A. J. (2010). AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of computational chemistry, 31(2), 455-461.

5. Abraham, M. J., Murtola, T., Schulz, R., Páll, S., Smith, J. C., Hess, B., & Lindahl, E. (2015). GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 1, 19-25.

6. Valdés-Tresanco, M.S., Valdés-Tresanco, M.E., Valiente, P.A. and Moreno E. gmx_MMPBSA: A New Tool to Perform End-State Free Energy Calculations with GROMACS. Journal of Chemical Theory and Computation, 2021 17 (10), 6281-6291. https://pubs.acs.org/doi/10.1021/acs.jctc.1c00645.

7. Bill R. Miller, T. Dwight McGee, Jason M. Swails, Nadine Homeyer, Holger Gohlke, and Adrian E. Roitberg. MMPBSA. py: An Efficient Program for End-State Free Energy Calculations. Journal of Chemical Theory and Computation, 2012 8 (9), 3314-3321. https://pubs.acs.org/doi/10.1021/ct300418h.