Sandbox Reserved 1845: Difference between revisions
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== Mutation Sites of Interest == | == Mutation Sites of Interest == | ||
To improve the catalytic activity and thermostability of LCC, Tournier et al. (2020) 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. | To improve the catalytic activity and thermostability of LCC, Tournier et al. (2020) 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. | ||
[[Image: | [[Image:Conserved amino acid.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. ]] | ||
From this screen, two mutations at Phe243 (F243I and F243W) were shown to improve catalytic activity by optimizing substrate positioning within the groove. To increase thermostability, the authors targeted a region of the enzyme that is structurally analogous to known divalent [https://en.wikipedia.org/wiki/Metal-binding_protein metal binding] sites in other cutinases. Instead of using stabilizing ions, which could complicate industrial degradation processes, they engineered a [https://en.wikipedia.org/wiki/Disulfide disulfide bridge] by mutating Asp238 and Ser283 to Cys residues (D238C/S283C). Additional mutations were selected based on thermostability screening. Among these, Y127G improved the melting point without reducing activity. | From this screen, two mutations at Phe243 (F243I and F243W) were shown to improve catalytic activity by optimizing substrate positioning within the groove. To increase thermostability, the authors targeted a region of the enzyme that is structurally analogous to known divalent [https://en.wikipedia.org/wiki/Metal-binding_protein metal binding] sites in other cutinases. Instead of using stabilizing ions, which could complicate industrial degradation processes, they engineered a [https://en.wikipedia.org/wiki/Disulfide disulfide bridge] by mutating Asp238 and Ser283 to Cys residues (D238C/S283C). Additional mutations were selected based on thermostability screening. Among these, Y127G improved the melting point without reducing activity. | ||
