Part:BBa_K2982003:Design
Coding sequence for W159H/S214H IsPETase double mutant
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 348
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 304
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 348
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 348
Illegal AgeI site found at 627 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The mutation site, W159 locate in substrate binding site, subsite II where three MHET moieties are bound through hydrophobic interaction. Trp 159 in PETase has effect of extending the hydrophobic surface adjacent to the active site. Mutation, W159H results in more space on binding center. The mutation site, S214 locate in the edge of the substrate binding site have effect on substrate binding. Both single mutant of W159H and S214H show higher activity than that of the wild type enzyme. Histidine is often found in the active sites of enzymes, where its imidazole ring can readily switch between uncharged or positively charged to catalyze the making and breaking of bonds. In TfCut2, Histidine 169 and Histidine 224 are located at the corresponding positions of Trpytophan 159 and Serine 214 in subsite II of IsPETase. The resulting double mutant may have effect on substrate binding.
Source
Modified from wild type IsPETase sequence from 'Characterization and engineering of a plastic-degrading aromatic polyesterase', PNAs, 2018.
References
Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115