Sandbox Reserved 1846: Difference between revisions

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== Mutation Sites of Interest ==

To improve the catalytic activity and thermostability of LCC, Tournier et al. used structure-guided enzyme engineering based on the crystal structure of LCC bound to a model PET substrate. Using [https://en.wikipedia.org/wiki/Docking_(molecular) molecular docking] and enzyme–substrate contact analysis, the researchers identified <scene name='10/1075247/Original_15_mutation_structure/5'>15 residues</scene> in the first contact shell surrounding the substrate-binding groove. Of these, 11 positions were selected for [https://en.wikipedia.org/wiki/Saturation_mutagenesis#:~:text=Saturation%20mutagenesis%2C%20or%20site%20saturation,amino%20acids%20at%20the%20position. saturation mutagenesis] to determine how mutations could affect PET depolymerization. These sites were chosen for their interactions with the PET-like ligand or their proximity to the active site. Highly conserved residues essential for catalysis or structural stability were excluded. ~~Using~~ [https://consurf.tau.ac.il/consurf_index.php. ConSurf] ~~demonstrates~~ the residues that ~~are~~ conserved ~~over~~ all ~~versions~~ of LCC~~, to determine what other variations have changed, seen in~~ Figure 3.

To improve the catalytic activity and thermostability of LCC, Tournier et al. used structure-guided enzyme engineering based on the crystal structure of LCC bound to a model PET substrate. Using [https://en.wikipedia.org/wiki/Docking_(molecular) molecular docking] and enzyme–substrate contact analysis, the researchers identified <scene name='10/1075247/Original_15_mutation_structure/5'>15 residues</scene> in the first contact shell surrounding the substrate-binding groove. Of these, 11 positions were selected for [https://en.wikipedia.org/wiki/Saturation_mutagenesis#:~:text=Saturation%20mutagenesis%2C%20or%20site%20saturation,amino%20acids%20at%20the%20position. saturation mutagenesis] to determine how mutations could affect PET depolymerization. These sites were chosen for their interactions with the PET-like ligand or their proximity to the active site. Highly conserved residues essential for catalysis or structural stability were excluded. [https://consurf.tau.ac.il/consurf_index.php. ConSurf] displays the residues that were conserved in all variants of LCC (Figure 3).

[[Image:Conserved amino acids.jpg|400 px|right|thumb|Figure 3: Image of protein structure, amino acids are colored depending on how often they are conserved in structure. Legend is included. ]]

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=== Ser 283 & Asp 238 ===

Ser 283 and Asp 238 are located <scene name='10/1075247/Start_material_for_s283_and_d2/2'>outside of the binding pocket</scene>. <scene name='10/1075247/S283-d238/5'>These wild-type residues</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/4ebo_disulfide_bond_skyblue/2'>S283C and D238C mutation</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.

Ser 283 and Asp 238 are located <scene name='10/1075247/Start_material_for_s283_and_d2/2'>outside of the binding pocket</scene>. <scene name='10/1075247/S283-d238/5'>These wild-type residues</scene> were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/4ebo_disulfide_bond_skyblue/2'>S283C and D238C mutation</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.

== Group Mutations ==

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@@ Line 34: / Line 34: @@
 == Mutation Sites of Interest ==
-To improve the catalytic activity and thermostability of LCC, Tournier et al. used structure-guided enzyme engineering based on the crystal structure of LCC bound to a model PET substrate. Using [https://en.wikipedia.org/wiki/Docking_(molecular) molecular docking] and enzyme–substrate contact analysis, the researchers identified <scene name='10/1075247/Original_15_mutation_structure/5'>15 residues</scene> in the first contact shell surrounding the substrate-binding groove. Of these, 11 positions were selected for [https://en.wikipedia.org/wiki/Saturation_mutagenesis#:~:text=Saturation%20mutagenesis%2C%20or%20site%20saturation,amino%20acids%20at%20the%20position. saturation mutagenesis] to determine how mutations could affect PET depolymerization. These sites were chosen for their interactions with the PET-like ligand or their proximity to the active site. Highly conserved residues essential for catalysis or structural stability were excluded. Using [https://consurf.tau.ac.il/consurf_index.php. ConSurf] demonstrates the residues that are conserved over all versions of LCC, to determine what other variations have changed, seen in Figure 3.
+To improve the catalytic activity and thermostability of LCC, Tournier et al. used structure-guided enzyme engineering based on the crystal structure of LCC bound to a model PET substrate. Using [https://en.wikipedia.org/wiki/Docking_(molecular) molecular docking] and enzyme–substrate contact analysis, the researchers identified <scene name='10/1075247/Original_15_mutation_structure/5'>15 residues</scene> in the first contact shell surrounding the substrate-binding groove. Of these, 11 positions were selected for [https://en.wikipedia.org/wiki/Saturation_mutagenesis#:~:text=Saturation%20mutagenesis%2C%20or%20site%20saturation,amino%20acids%20at%20the%20position. saturation mutagenesis] to determine how mutations could affect PET depolymerization. These sites were chosen for their interactions with the PET-like ligand or their proximity to the active site. Highly conserved residues essential for catalysis or structural stability were excluded. [https://consurf.tau.ac.il/consurf_index.php. ConSurf] displays the residues that were conserved in all variants of LCC (Figure 3).
 [[Image:Conserved amino acids.jpg|400 px|right|thumb|Figure 3: Image of protein structure, amino acids are colored depending on how often they are conserved in structure. Legend is included. ]]
@@ Line 46: / Line 46: @@
 === Ser 283 & Asp 238 ===
-Ser 283 and Asp 238 are located <scene name='10/1075247/Start_material_for_s283_and_d2/2'>outside of the binding pocket</scene>. <scene name='10/1075247/S283-d238/5'>These wild-type residues</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/4ebo_disulfide_bond_skyblue/2'>S283C and D238C mutation</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.
+Ser 283 and Asp 238 are located <scene name='10/1075247/Start_material_for_s283_and_d2/2'>outside of the binding pocket</scene>. <scene name='10/1075247/S283-d238/5'>These wild-type residues</scene> were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/4ebo_disulfide_bond_skyblue/2'>S283C and D238C mutation</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.
 == Group Mutations ==