Part:BBa_K3468022
PETase Key g The PETase is an enzyme, which can hydrolyze PET and this mutation protein is changed on the basis of the PETase.This mutant is more stable in higher temperature compared with the wild type.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 240
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 240
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 240
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 240
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 240
- 1000COMPATIBLE WITH RFC[1000]
Disulfide bond plays a significant role in the thermal stability of proteins. The substitution of cysteine for alanine in A202C mutation and the substitution of cysteine for glutamic acid in E231C conferred a new disulfide bond
In addition to electrostatic effects, hydrophobic interactions have a marked influence on enzymatic performance. Q127L prompts the hydrophobic packing in the protein interior
The indolyl of S54W formed a offset parallel π-π stacking interaction with the phenolic group of the Y69.
For the Q119D mutation, the substitution of asparate for glutamine in Q119D, mutation conferred new hydrogen bonds with the aminogroup of Ser121 and Ser122. The substitution of arginine to asparagine in N114R formed new salt bridges with D118.
The substitution of arginine to asparagine in N73R and the substitution of glutamate to asparagine in N72E prompted new salt bridges between the oxhydryl of T77E and the amino of R34.
The substitution of proline for threonline in T266P mutation reduced the conformational entropy of the local loop region.
The phenyl group of S214F formed a offset parallel π-π stacking interaction with the indolyl of the W185.
The substitution of proline for asparagine in N275P mutation reduced the conformational entropy of the local loop region
For the S115R mutation, the substitution of arginine for serine in S115R , conferred new hydrogen bonds with the carboxyl of A114 and A118
In order to select the mutants with the best thermostability,molecular dynamics (MD) simulation analyses were conducted.In our project, we use GROMACS, which is one of the most popular MD software packages for protein, lipid, and nucleic acid.
We conducted MD simulations, of wild type PETase (WT) and its eight progressively thermostable mutants , performed at 343K.The root mean square deviation (RMSD) ,the root mean square fluctuation(RMSF),radius of gyration(Rg),solvent accessible surface area(SASA),free-energy landscape and number of hydrogen bond analyses reproduced the correct trends in stability changes of the wild-type and mutated PETase.
Here, we named it as number 12.
Many previous studies suggested that proteins with lower RMSD values compared with the starting structure tend to be more thermostable during MD simulation. The RMSD of our mutant is lower than the wild-type, and the fluctuation amplitude is smaller, wh
Mutants with lower RMSF values during MD simulation tend to be more thermostable. Except for mutant our , other RMSF plots are very similar with wild type PETase and are not indicative of stability at 343K. But the RMSF value of mutant our is significantly lower than that of wild type.
Protein folding is driven by hydrophobic effect and is temperature dependent . Under normal conditions, the hydrophilic residues are usually on the protein surface, while hydrophobic residues are generally buried inside the protein away from the aqueous environment . If protein denaturation occurs, the hydrophobic region will be exposed to the solvent.The SASA values of our mutant is lower than that of the wild-type, which indicates that our mutant is more stable than wild-type PETase at 343K.
The hydrogen bond is another important temperature-dependent interaction in maintaining the stability of protein.The higher number of intramolecular hydrogen bonds conferred mutants have higher resistant against heat denaturation. Except for mutant our and 12M, the number of intramolecular hydrogen bonds in other mutants are very similar with wild type PETase. In contrast, the higher number of intramolecular hydrogen bonds present in mutant our implies they have higher thermostability.
The compactness of protein is another indicator to measure the stability of protein. The effect of temperature to the overall dimension of PETaes was gleaned from the Rg analysis. Overall, the Rg of our mutant remaines constant throughout the simulation which implies that the mutant structure is capable to maintain its original compactness as the temperature rises.
The mutant our shows the required flexibility at appropriate temperature ranges and maintain conformational stability at high temperature. It shows a deep and rugged free-energy landscape,which indicates mutant our has more possibilities to have higher thermostability. ich indicates that our mutant is more stable than wild-type PETase at 343K.
The benzene ring is located in a gully between two aromatic residues TYR87 and TRP185. The distance of PI-PI bond is 4.8A between TRP185 and the benzene ring in part of the first MHET. The whole ligand just fits into the pocket and binds tightly. Three residues of Ser160, Asp206 and His237 formed a catalytic triplet, and ranging was performed: Y87:4.7A ;M161:5.2A; H237:4.8A.
Significance of molecular docking for mutant work: The molecular docking model can predict the binding mode and affinity of mutants and ligands, and promote the work of mutants by analyzing the result model.
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