Overview

This project, we investigated 3 different mutation of T7-IsPETase, T7-IsPETase^Thr116Ala, T7-IsPETase^{Thr116Ala/Met154Thr}, T7-IsPETase^{Thr116Ala/Lys259Glu}. In a meeting with Dr. Huang, he suggested primarily how we could conduct dry lab experimentation to predict our mutated enzyme activity. The mutations in our enzymes are new and unexplored by current research, which increases the need to use dry lab predictions (See Integrated HP). Dr. Huang suggested that we can use visualization software to predict where our mutations are in terms of the enzyme and offered to provide us with the software. In this case, we decided to use Chimera for structure visualization and AutoDock Vina for docking.

methodology

To explore this further, we downloaded UCSF Chimera with autodock vina installed in order to visualize the PDB file and dock the enzyme with ligand. We downloaded the PDB file from RCSB PDB for our wild-type T7-IsPETase (5XJH). Chimera is an extensible program for interactive visualization of molecular structures and related data, including docking results. AutoDock Vina is one of the extensions that works with Chimera for docking. With the docking technology, we are able to see how the wildtype and mutated PETase binds with MHET.

First, we use the predicted structural features in Chimera to visualize the structure of T7-IsPETase. We find the amino acids we are mutating, Thr116, Met154, and Lys259, and show the atoms structure in that certain area.

Figure 1: Ribbons colored in Red are Thr116, Met154, and Lys259 as labeled.

Next, we mutated the sequence of T7-IsPETase inside Chimera. We have 3 different mutations, Thr116Ala, Thr116Ala/Met154Thr, and Thr116Ala/Lys259Glu.

3D Structure

1. T7-IsPETase^Thr116Ala
   
   In this mutation, the threonine at position 116, which has a polar uncharged side chain, is replaced by alanine, a hydrophobic amino acid. This shift alters the interaction between residue 116 and the substrate, potentially affecting the enzyme’s catalytic function.



Figure 2: T7-IsPETase^Thr116Ala BBa_K5094003

2. T7-IsPETase^{Thr116Ala/Met154Thr}
   
   This double mutation involves the substitution of threonine for alanine at position 116 and methionine for threonine at position 154. These changes introduce a hydrophobic-to-polar shift in one residue and the reverse in another, impacting both amino acids' properties and interactions with the substrate.



Figure 3: T7-IsPETase^{Thr116Ala/Met154Thr} BBa_K5094004

3. T7-IsPETase^{Thr116Ala/Lys259Glu}
   
   In this double mutation, threonine is replaced by alanine at position 116, and lysine (a positively charged amino acid) is swapped for glutamic acid (negatively charged) at position 259. This combination modifies the charge and hydrophobicity of key regions in the enzyme, likely altering both its binding affinity and catalytic properties.



Figure 4: T7-IsPETase^{Thr116Ala/Lys259Glu} BBa_K5094005

To further explore these mutations, Dr. Huang suggested using AutoDock Vina to dock MHET oligomers with the enzymes. For this purpose, we added the following structures to Chimera for binding simulations:

1. 2HE-(MHET)4 [1]

Docking

1. T7-IsPETase

   Figure 5: WildtypeT7-IsPETase Binding with 2HE-(MHET)4

   The docking analysis was performed using AutoDock Vina to explore the binding interactions between 2HE-(MHET)4 and the wildtype T7-IsPETase (5XJH) enzyme. T7-IsPETase consists of 290 amino acids, with several key substrate binding sites (I-IV) and an active site critical for enzymatic function. The substrate binding residues were identified as follows:

   1. Tyr87, Met161, Trp185
   2. Ser92, Ser98, Ser242
   3. Ser242, Gly243, Gln247
   4. Ser245, Gln247, Ala248

   The active sites are Ser160, Asp206, and His237 amino acids as observed from docking and as shown in research[2]. Ser160, Asp206 and His237 will form a catalytic triad in the oxyanion hole to perform hydrolysis intermediate of 2-HE-(MHET)4.

   Steric contacts between the ligand and receptor were analyzed to evaluate whether the ligand is positioned favorably within the enzyme's binding site. A total of 19+11(2)+14+16 steric contacts were identified between the ligand and nearby residues, which subsite I, contains 19, subsite II contains 11(2), subsite III contains 14, subsite IV contains 16. These steric contacts were shown in the yellow line in the chimera.

Using Chimera docking visualize tool and Hydrogen Bond tool, hydrogen bond between ligand and receptor are observed in following amino acids:

1. Ala248[2.269 Å]
2. Gln247 [2.430 Å]
3. Tyr87 [2.451 Å]

The Hydrogen bonds between the ligand and residues Ala248, Gln247, and Tyr87 suggest strong interactions that help stabilize the substrate in the enzyme’s binding pocket. These support proper orientation for enzymatic hydrolysis.

2. T7-IsPETase^Thr116Ala

   Figure 6: T7-IsPETase^Thr116Ala Binding with 2HE-(MHET)4

   Figure 6 is T7-IsPETase^Thr116Ala, consisting of 290 amino acids as 5XJH with Thr116 mutated, with 1 key substrate binding sites and an active site critical for enzymatic function. The substrate binding amino acids were following:

   1. Ser160, Asp206, His237

   The active sites of wild type T7-IsPETase, Ser160, Asp206, and His237 amino acids match the steric contacts between MHET and T7-IsPETase^Thr116Ala. As a result, we predict this is also the active site of T7-IsPETase^Thr116Ala.

   A total of 23 steric contacts were identified between the ligand and nearby residues, which mainly contacts to the active sites of wild type T7-IsPETase These steric contacts were shown in the yellow line in the chimera.

Using Chimera docking visualize tool and Hydrogen Bond tool, hydrogen bond between ligand and receptor are observed in following amino acids:

1. Ser245[2.194 Å]

In the T7-IsPETase^Thr116Ala, the key catalytic triad of Ser160, Asp206, and His237 is preserved, similar to the wild-type enzyme. The conservation of these active site residues suggests that the mutant enzyme retains its ability to catalyze hydrolysis, similar to the wild-type enzyme. Also, the docking result shows that the mutant Thr116Ala does not play a significant role in binding the ligand to the receptor. While it doesn’t play a significant role in the binding, from the docking result we can see that it does affect the overall binding structure. As the figure shows, ligand shows a different orientation while binding to the receptor and main steric contacts zone are within Ser160, Aso206, and His237 3 amino acids. Also, the amount of hydrogen bonds has decreased from 3 to 1.

3. T7-IsPETase^{Thr116Ala/Met154Thr}

   Figure 7: T7-IsPETase^{Thr116Ala/Met154Thr} Binding with 2HE-(MHET)4

   Figure 7 is T7-IsPETase^{Thr116Ala/Met154Thr}, consisting of 290 amino acids as 5XJH with Thr116 and Met154 mutated. Same as wildtype, with 4 key substrate binding sites (I-IV)and an active site critical for enzymatic function. The substrate binding amino acids were following:

   1. Tyr87, Thr88, Gly80, Ser260, His227
   2. Ser92, Ser93, Ser242
   3. Trp98, Ser242, Gly243
   4. Ala45, Ser46, Ala246

   The active site of wildtype T7-IsPETase, Ser260 and His227 amino acids, matches the steric contacts of between MHET and T7-IsPETase^{Thr116Ala/Met154Thr}. As a result, we predict this is also the active site of T7-IsPETase^{Thr116Ala/Met154Thr}.

   A total of 71 steric contacts were identified between the ligand and nearby residues, which subsite I contains 21, II contains 19, III contains 17, IV contains 15 and some contacts to the active sites of wild type T7-IsPETase These steric contacts were shown in the yellow line in the chimera.

Using Chimera docking visualize tool and Hydrogen Bond tool, hydrogen bond between ligand and receptor are observed in following amino acids:

1. Gly80[2.472 Å]
2. Ser260[2.447 Å]

In the T7-IsPETase^Thr116Ala, the key catalytic triad of Ser160, Asp206, and His237 is also preserved, similar to the wild-type enzyme. The conservation of these active site residues suggests that the mutant enzyme still has its ability to function similar to the wild-type enzyme. Also, the docking result shows that the mutant Thr116Ala/Met154Thr has more similarity with wildtype compared to Thr116 and it has more steric contacts than wild type T7-IsPETase. Though it isn’t significant, from the docking result we predict it does affect the binding structure and can somehow boost the enzyme kinetics. Also, as the figure shows, the orientation of the ligand is presenting the same orientation of the wild-type docking result and contains 2 hydrogen bonds with receptors. One of the hydrogen bonds binds with Ser260, which is one of the active sites, suggesting this mutation can increase the enzyme kinetics and increase proficiency.

4. T7-IsPETase^{Thr116Ala/Lys259Glu}

   Figure 8: T7-IsPETase^{Thr116Ala/Lys259Glu} Binding with 2HE-(MHET)4

   Figure 8 is T7-IsPETase^{Thr116Ala/Lys259Glu}, consisting of 290 amino acids as 5XJH with Thr116 and Lys259 mutated. Same as wildtype, with 4 key substrate binding sites (I-IV)and an active site critical for enzymatic function. The substrate binding amino acids were following:

   1. Gly86, Tyr87, Thr88, Ser160, Ile208, His237
   2. Thr88, Ser92, Ser93
   3. Ser92, Gly243, Gln247
   4. Gln247, Ser245, Ala248, Asn246

   The active site of wildtype T7-IsPETase, Ser260 and His227 amino acid, matches the steric contacts of between MHET and T7-IsPETase^{Thr116Ala/Lys259Glu}. As a result, we predict this is also the active site of T7-IsPETase^{Thr116Ala/Lys259Glu}.

   A total of 58 steric contacts were identified between the ligand and nearby residues, which subsite I contains 24, II contains 9, III contains 2, IV contains 23 and some contacts to the active sites of wild type T7-IsPETase These steric contacts were shown in the yellow line in the chimera.

In the T7-IsPETase^{Thr116Ala/Lys259Glu}, the key catalytic triad of Ser160, Asp206, and His237 is also preserved, and the binding is similar to T7-IsPETase^{Thr116Ala/Met154Thr}. The conservation of these active site residues suggests that the mutant enzyme can function similar to the wild-type enzyme. Also, the docking result shows that the mutant Thr116Ala/Lys259Glu has more similarity with wildtype compared to Thr116 but less than Thr116Ala/Met154Thr and it has less steric contacts than wild type T7-IsPETase. Though it isn’t significant, from the docking result we can predict that it does affect the binding structure and causes restricted kinetics due to the short number of steric contacts. Also, as the figure shows, the orientation of the ligand is presenting the same orientation of the wild-type docking result and contains 2 hydrogen bonds with receptors. One of the hydrogen bonds binds with Ser260, which is one of the active sites, suggesting this mutation can bind with the active sites with enzyme functioning.

1. Ser160[2.859 Å]
2. Ser245[2.104 Å]

The Hydrogen bonds between the ligand and residues Ala248, Gln247, and Tyr87 suggest strong interactions that help stabilize the substrate in the enzyme’s binding pocket. These support proper orientation for enzymatic hydrolysis.

Citations

National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 130323426, Linear pet tetramer + EG, 1ST series. Retrieved October 2, 2024 from https://pubchem.ncbi.nlm.nih.gov/compound/Linear-pet-tetramer-_-EG_-1ST-series.

Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation - Scientific Figure on ResearchGate. https://www.researchgate.net/figure/Active-site-of-IsPETase-a-Catalytic-triad-and-the-docking-model-of-the-reaction_fig1_322723916 [accessed 2 Oct 2024]