Sandbox Reserved 1846: Difference between revisions

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== Structural Overview ==

LCC consists of one domain. <scene name='10/1075246/4eb0_helix_sheet/2'>Alpha helices and beta sheets</scene> are interspersed throughout the protein, with beta sheets (yellow) forming a stable central core surrounded by alpha helices (magenta) that contribute to the overall folding. This creates a predominantly α/β hydrolase fold that is typical of cutinases.

LCC consists of one domain. <scene name='10/1075246/4eb0_helix_sheet/4'>Alpha helices and beta sheets</scene> are interspersed throughout the protein, with beta sheets (yellow) forming a stable central core surrounded by alpha helices (magenta) that contribute to the overall folding. This creates a predominantly α/β hydrolase fold that is typical of cutinases.

=== Catalytic Triad ===

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=== Ligand Binding Pocket ===

The <scene name='10/1075246/4eb0_with_colored_ligand_stick/2'>substrate-binding site</scene> of LCC is a long, mainly hydrophobic groove that accommodates PET chains. This groove includes three subsites—designated −2, −1, and +1—that interact with specific PET units near the scissile ester bond. Hydrophobic residues such as F125, V212, M166, and F243 line the groove and facilitate binding by interacting with the [https://en.wikipedia.org/wiki/Aromatic_compound aromatic] rings of the PET molecule. These interactions help align the substrate in the correct position for catalysis.

The <scene name='10/1075246/4eb0_with_colored_ligand_stick/3'>substrate-binding site</scene> of LCC is a long, mainly hydrophobic groove that accommodates PET chains. This groove includes three subsites—designated −2, −1, and +1—that interact with specific PET units near the scissile ester bond. Hydrophobic residues such as F125, V212, M166, and F243 line the groove and facilitate binding by interacting with the [https://en.wikipedia.org/wiki/Aromatic_compound aromatic] rings of the PET molecule. These interactions help align the substrate in the correct position for catalysis.

The <scene name='10/1075246/4eb0_surface_w_stick_ligand/1'>molecular surface view of the enzyme-ligand interaction</scene> shows the overall shape and depth of the binding groove. The <scene name='10/1075246/4eb0_hydrophobicity_ligand/1'>cartoon representation of the enzyme-ligand interaction</scene>, the enzyme is shown as a ribbon diagram with the hydrophobic residues colored pink, to show how the PET chain fits snugly into the groove.

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=== S283 & D238 ===

Two wild-type residues, <scene name='10/1075247/S283-d238/5'>S283 and D238</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='10/1075246/C283-c238/1'>S283C and D238C mutant</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.

Two wild-type residues, <scene name='10/1075247/S283-d238/5'>S283 and D238</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='10/1075246/C283-c238/2'>S283C and D238C mutant</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.

</StructureSection>

Sandbox Reserved 1846: Difference between revisions

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@@ Line 17: / Line 17: @@
 == Structural Overview ==
-LCC consists of one domain. <scene name='10/1075246/4eb0_helix_sheet/2'>Alpha helices and beta sheets</scene> are interspersed throughout the protein, with beta sheets (yellow) forming a stable central core surrounded by alpha helices (magenta) that contribute to the overall folding. This creates a predominantly α/β hydrolase fold that is typical of cutinases.
+LCC consists of one domain. <scene name='10/1075246/4eb0_helix_sheet/4'>Alpha helices and beta sheets</scene> are interspersed throughout the protein, with beta sheets (yellow) forming a stable central core surrounded by alpha helices (magenta) that contribute to the overall folding. This creates a predominantly α/β hydrolase fold that is typical of cutinases.
 === Catalytic Triad ===
@@ Line 25: / Line 25: @@
 === Ligand Binding Pocket ===
-The <scene name='10/1075246/4eb0_with_colored_ligand_stick/2'>substrate-binding site</scene> of LCC is a long, mainly hydrophobic groove that accommodates PET chains. This groove includes three subsites—designated −2, −1, and +1—that interact with specific PET units near the scissile ester bond. Hydrophobic residues such as F125, V212, M166, and F243 line the groove and facilitate binding by interacting with the [https://en.wikipedia.org/wiki/Aromatic_compound aromatic] rings of the PET molecule. These interactions help align the substrate in the correct position for catalysis.
+The <scene name='10/1075246/4eb0_with_colored_ligand_stick/3'>substrate-binding site</scene> of LCC is a long, mainly hydrophobic groove that accommodates PET chains. This groove includes three subsites—designated −2, −1, and +1—that interact with specific PET units near the scissile ester bond. Hydrophobic residues such as F125, V212, M166, and F243 line the groove and facilitate binding by interacting with the [https://en.wikipedia.org/wiki/Aromatic_compound aromatic] rings of the PET molecule. These interactions help align the substrate in the correct position for catalysis.
 The <scene name='10/1075246/4eb0_surface_w_stick_ligand/1'>molecular surface view of the enzyme-ligand interaction</scene> shows the overall shape and depth of the binding groove. The <scene name='10/1075246/4eb0_hydrophobicity_ligand/1'>cartoon representation of the enzyme-ligand interaction</scene>, the enzyme is shown as a ribbon diagram with the hydrophobic residues colored pink, to show how the PET chain fits snugly into the groove.
@@ Line 48: / Line 48: @@
 === S283 & D238 ===
-Two wild-type residues, <scene name='10/1075247/S283-d238/5'>S283 and D238</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='10/1075246/C283-c238/1'>S283C and D238C mutant</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.
+Two wild-type residues, <scene name='10/1075247/S283-d238/5'>S283 and D238</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='10/1075246/C283-c238/2'>S283C and D238C mutant</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.
 </StructureSection>