{"doi":"10.1101/2020.03.19.999235","title":"Evolution of an enzyme conformational ensemble guides design of an efficient biocatalyst","abstract":"Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we used room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 ( k cat / K M 160 M −1 s −1 ). We observed that catalytic residues were increasingly rigidified, the active site became better pre-organized, and its entrance was widened. Based on these observations, we engineered HG4, an efficient biocatalyst ( k cat / K M 120,000 M −1 s −1 ) containing active-site mutations found during evolution but not distal ones. HG4 structures revealed that its active site was pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":119696,"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":16,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.954,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":267622,"name":"Rojo V. Rakotoharisoa","orcid":"0000-0002-6901-4529","position":1,"is_corresponding":false},{"id":227692,"name":"Michael C. Thompson","orcid":"0000-0002-6099-2027","position":2,"is_corresponding":false},{"id":267623,"name":"Niayesh Zarifi","orcid":"0000-0003-4748-7082","position":3,"is_corresponding":false},{"id":269363,"name":"Erin Nguyen","orcid":null,"position":4,"is_corresponding":false},{"id":269364,"name":"Nurzhan Mukhametzhanov","orcid":null,"position":5,"is_corresponding":false},{"id":555651,"name":"Lin Liu","orcid":"0000-0001-7982-6005","position":6,"is_corresponding":false},{"id":63530,"name":"James S. Fraser","orcid":"0000-0002-5080-2859","position":7,"is_corresponding":false},{"id":267625,"name":"Roberto A. Chica","orcid":"0000-0003-3789-9841","position":8,"is_corresponding":false},{"id":267621,"name":"Aron Broom","orcid":"0000-0001-8285-156X","position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":null,"created_at":"2026-07-18T23:14:13.002105Z","pmid":null,"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":[]}