{"doi":"10.1002/pro.4997","title":"Understanding the stability of a plastic‐degrading Rieske iron oxidoreductase system","abstract":"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 (TPA DO PDB ID 7Q05 ) is a structurally characterized heterohexameric α 3 β 3 RO that, with its cognate reductase (TPA RED ), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPA DO /TPA RED 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 DO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature ( T m ) of 39.9°C for the monomeric TPA RED , while the independent T m of TPA DO is 50.8°C. Differential scanning calorimetry revealed a two‐step thermal decomposition pathway for TPA DO with T m values of 47.6 and 58.0°C (Δ H = 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 TPA DO 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 DO stability, we propose prioritizing the re‐engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.","journal":"Protein Science","year":2024,"id":463252,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":6,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.957,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1066935,"name":"Anjani K. Maurya","orcid":"0000-0002-9134-6693","position":1,"is_corresponding":false},{"id":1293453,"name":"Ronivaldo Rodrigues da Silva","orcid":"0000-0002-6504-8406","position":2,"is_corresponding":false},{"id":1293454,"name":"Emmanuel Akpoto","orcid":"0000-0003-2413-6554","position":3,"is_corresponding":false},{"id":1293455,"name":"Arun S. Asundi","orcid":"0000-0003-0333-6784","position":4,"is_corresponding":false},{"id":1293759,"name":"Julia Fecko","orcid":null,"position":5,"is_corresponding":false},{"id":274208,"name":"Neela H. Yennawar","orcid":"0000-0001-7278-659X","position":6,"is_corresponding":false},{"id":780547,"name":"Ritimukta Sarangi","orcid":"0000-0002-2764-2279","position":7,"is_corresponding":false},{"id":761170,"name":"Christopher J. Tassone","orcid":"0000-0002-6664-2554","position":8,"is_corresponding":false},{"id":455401,"name":"Thomas Weiß","orcid":"0000-0002-4991-6779","position":9,"is_corresponding":false},{"id":498167,"name":"Jennifer L. DuBois","orcid":"0000-0001-5593-3391","position":10,"is_corresponding":false},{"id":861658,"name":"Jessica Lusty Beech","orcid":"0000-0002-4212-6133","position":0,"is_corresponding":true}],"reference_count":84,"raw_metadata":null,"created_at":"2026-07-19T02:04:28.838123Z","pmid":"38723110","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}