{"doi":"10.1016/j.jbc.2024.105783","title":"Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model","abstract":"Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.","journal":"Journal of Biological Chemistry","year":2024,"id":420583,"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":33,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9619,"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":1212036,"name":"Ziyue Dong","orcid":"0000-0001-8332-9212","position":1,"is_corresponding":false},{"id":1212037,"name":"Christopher T. Canova","orcid":"0000-0003-2914-8052","position":2,"is_corresponding":false},{"id":1212038,"name":"Francesco Destro","orcid":"0000-0002-0977-0672","position":3,"is_corresponding":false},{"id":1212039,"name":"Marina Cañellas","orcid":"0000-0002-7107-191X","position":4,"is_corresponding":false},{"id":1212040,"name":"Mikaila C. Hoffman","orcid":"0000-0001-7879-7572","position":5,"is_corresponding":false},{"id":1212041,"name":"Jeanne Maréchal","orcid":"0009-0003-5318-3082","position":6,"is_corresponding":false},{"id":1212042,"name":"T. M. Johnson","orcid":"0000-0001-9303-2949","position":7,"is_corresponding":false},{"id":1212712,"name":"Maya Zheng","orcid":null,"position":8,"is_corresponding":false},{"id":494039,"name":"Gabriela S. Schlau‐Cohen","orcid":"0000-0001-7746-2981","position":9,"is_corresponding":false},{"id":1212713,"name":"Maria Fátima Lucas","orcid":null,"position":10,"is_corresponding":false},{"id":1212043,"name":"Richard D. Braatz","orcid":"0000-0003-4304-3484","position":11,"is_corresponding":false},{"id":678135,"name":"Kayla G. Sprenger","orcid":"0000-0002-7505-7920","position":12,"is_corresponding":false},{"id":267216,"name":"Christopher A. Voigt","orcid":"0000-0003-0844-4776","position":13,"is_corresponding":false},{"id":923989,"name":"Anthony J. Sinskey","orcid":"0000-0002-1015-1270","position":14,"is_corresponding":false},{"id":1212035,"name":"En Ze Linda Zhong-Johnson","orcid":"0000-0002-0373-8399","position":0,"is_corresponding":true}],"reference_count":71,"raw_metadata":null,"created_at":"2026-07-19T01:57:31.041511Z","pmid":"38395309","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":[]}