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Bioinformatics, Computational and Systems Biology

Bioinformatics, Computational and Systems Biology - Poster Session D

Poster A7 - Optimizing Laccase Enzyme for Polyethylene Degradation Using Molecular Dynamics Analysis

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Plastic pollution continues to remain an environmental threat to our planet. In particular, Polyethylene (PE) found in plastic bags, is made up of long carbon-carbon chain polymers that are difficult to decompose naturally, making PE a leading contributor of pollution. Mycoremediation, a subset of bioremediation that uses the decomposition abilities of fungi to break down harmful pollutants, has been a potential solution to the problem of excess PE plastic waste.

Pleurotus Ostreatus, known commonly as oyster mushroom, have been shown to be particularly efficient in PE degradation as they produce oxidative enzymes that can catalyze the breakdown of PE polymers. One of the enzymes in oyster mushrooms is the laccase enzyme, coded for by the POX2 gene, which can function independently and catalyze the oxidation of several phenolic and non-phenolic compounds, including PE. To fully maximize the potential implementation and optimization of this enzyme in the degradation of polyethylene, it is necessary to characterize the physical properties of the protein in its wild-type and mutated forms. To achieve this, molecular dynamics simulations were performed and analyzed.
Using NAMD and Visual Molecular Dynamics (VMD), this study tests different conditions to determine the stability and efficiency of the laccase enzyme in degrading PE by providing an atomic-level understanding of the thermal stability of our wild-type enzyme vs. mutated enzyme. The results of this study hope to inform potential strategies in the optimization of laccase stability.

Materials and

Methods:  To perform the simulations, the PDB file for the wild-type structure of POX2 was obtained through the UniProt database, using its AlphaFold plugin tool. The PDB file was then loaded into VMD, alongside the necessary topology/parameter files that were sourced from CHARMM-GUI. The protein was solvated in a water box and equilibrated before finally running the NAMD simulation using the CHARMM36 force field. A single NAMD configuration file with the same parameters except for temperature was used for each simulation to avoid undesired variation in simulation conditions. The simulation was run for 10 nanoseconds at temperatures of 300K, 310K, 340K and 400K. Each simulation was minimized for 2000 steps, and Langevin dynamics were used to maintain constant temperature. Once these simulations were complete, a variety of analysis tools including RMSD, RMSF, H-bond count and Ramachandran plot were used to characterize the behavior of the protein at increasing temperatures. RMSD values were each generated using the post-simulation analysis tools provided by VMD. The formula for RMSD values is shown in Figure 1, where Natoms is the number of atoms in the analysis, and ri(t) is the position of atom i at time t.

Mutation libraries were generated using FireProt, a web-based server that automates the design of thermostable mutants, and mutations with the lowest FoldX (ΔΔG) values were selected for further experimentation. Finally, PDB structures for thermostable mutants were generated using ChimeraX’s AlphaFold Google Collab program.

Results, Conclusions, and Discussions: The results of the RMSD analysis of the wild type laccase enzyme at each temperature are shown in Figure 2.

Simulations of 300K, 310K and 340K, the RMSD values tend to plateau at ~2 Angstroms, indicating successful equilibration and relative stability. At 400K, RMSD values continuously trend upwards, exceeding 3 Angstroms, indicating reduced structural stability and potential loss of function. Further simulations for the 400K simulation were not conducted due to uncertainty about the required length.

Using FireProt, a library of 16 mutations were generated, of which 6 were correlated with FoldX values of less than -0.5, as shown in Figure 3.
The mutation correlated with the lowest FoldX value, E409P, was then implemented in the laccase amino acid sequence. ChimeraX’s AlphaFold Google Collab program then ran 5 predictions, saving the best as a PDB coordinate file. The IDDT values for each prediction are displayed in Figure 4.
A NAMD simulation of the mutant enzyme was then conducted at 400K. The RMSD analysis of the simulation was compared with the results of the wild type enzyme as shown in Figure 5.

The RMSD data suggests that the mutant laccase enzyme was able to equilibrate and converge successfully to a value of ~2 Angstroms at 400K, closely following the trajectory of the wild type enzyme at 300K.

After analyzing the effects of temperature on the stability of the protein, these effects were compared to those on a modified protein with a mutation calculated to maximize stability. The single mutation (E409P) showed promising results. The RMSD chart depicted deviations at 400K comparable to wild type deviations at 300K, a temperature at which the protein is considered stable. These results based on the simulation are not conclusive and further lab work will need to be performed to verify them.

Based on the results, a further analysis of the mutated protein can be conducted to verify the stability properties hypothesized during the simulation. This may have future implications in industrial applications of laccase, as the mutated gene would be functional in higher temperature environments usually present in bioremediation efforts.

Acknowledgements (Optional):