Sandbox Reserved 1845: Difference between revisions
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=== S283 & D238 === | === 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='10/1075248/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. | 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='10/1075248/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. | ||
== Group Mutations == | |||
These mutations were combined to create multi-mutant LCC variants with improved activity and thermostability. The two most successful variants were: | |||
ICCG: F243I / D238C / S283C / Y127G | |||
WCCG: F243W / D238C / S283C / Y127G | |||
Other stabilizing mutations, such as T96M, N246D, and N246M, were also tested but are not included in this page's protein model. These were excluded because they were not part of the top-performing mutant (ICCG), and therefore omitted for clarity.<ref name="Tournier"/> | |||
</StructureSection> | </StructureSection> | ||
