{"doi":"10.1101/2024.04.18.590141","title":"Computational stabilization of a non-heme iron enzyme enables efficient evolution of new function","abstract":"Directed evolution has emerged as a powerful tool for engineering new biocatalysts. However, introducing new catalytic residues can be destabilizing, and it is generally beneficial to start with a stable enzyme parent. Here we show that the deep learning-based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially-relevant non-native functions. For the Fe(II)/αKG enzyme tP4H, we performed site-saturation mutagenesis with both the wild-type and stabilized design variant and screened for activity increases in a non-native C-H hydroxylation reaction. We observed substantially larger increases in non-native activity for variants obtained from the stabilized scaffold compared to those from the wild-type enzyme. ProteinMPNN is user-friendly and widely-accessible, and straightforward structural criteria were sufficient to obtain stabilized, catalytically-functional variants of the Fe(II)/αKG enzymes tP4H and GriE. Our work suggests that stabilization by computational sequence redesign could be routinely implemented as a first step in directed evolution campaigns for novel biocatalysts.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":490719,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9486,"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":1005691,"name":"Kiera H. Sumida","orcid":"0000-0003-2773-9676","position":1,"is_corresponding":false},{"id":1225053,"name":"Jessica L. Caruso","orcid":"0009-0009-1692-3233","position":2,"is_corresponding":false},{"id":105706,"name":"David Baker","orcid":"0000-0001-7896-6217","position":3,"is_corresponding":false},{"id":499768,"name":"Jesse G. Zalatan","orcid":"0000-0002-1458-0654","position":4,"is_corresponding":false},{"id":1225052,"name":"Brianne R. King","orcid":"0000-0003-3592-5882","position":0,"is_corresponding":true}],"reference_count":42,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:08:37.162700Z","pmid":"39091854","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":[]}