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Understanding the stability of a plastic‐degrading Rieske iron oxidoreductase system.
- Source :
- Protein Science: A Publication of the Protein Society; Jun2024, Vol. 33 Issue 6, p1-19, 19p
- Publication Year :
- 2024
-
Abstract
- Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3β3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two‐step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol−1, respectively) for each step. Temperature‐dependent small‐angle x‐ray scattering and dynamic light scattering both detected heat‐induced dissociation of TPADO subunits at 53.8°C, followed by higher‐temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by β‐β interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re‐engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 09618368
- Volume :
- 33
- Issue :
- 6
- Database :
- Complementary Index
- Journal :
- Protein Science: A Publication of the Protein Society
- Publication Type :
- Academic Journal
- Accession number :
- 177562498
- Full Text :
- https://doi.org/10.1002/pro.4997