Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system.

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 (TPA PDB ID 7Q05) is a structurally characterized heterohexameric αβ RO that, with its cognate reductase (TPA), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPA/TPA 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 TPA through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (T) of 39.9°C for the monomeric TPA, while the independent T of TPA is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPA with T values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPA 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 TPA stability, we propose prioritizing the re-engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.