{"doi":"10.3389/fchem.2024.1415644","title":"Nα-acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification","abstract":"Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N ɑ -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5 , N 5 -di-methyl N α -acetyl-L-ornithine (k cat /K m = 7.32 ± 0.94 × 10 4 M −1 s −1 ). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC 50 = 58.7 μM; K i = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC 50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril K i = 35.9 ± 5.1 μM). The active site structure of di-Zn Ec ArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).","journal":"Frontiers in Chemistry","year":2024,"id":500724,"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":0,"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":553066,"name":"J. Osipiuk","orcid":null,"position":1,"is_corresponding":false},{"id":1349947,"name":"Małgorzata Korbas","orcid":"0000-0003-3064-0132","position":2,"is_corresponding":false},{"id":234152,"name":"M. Endres","orcid":null,"position":3,"is_corresponding":false},{"id":1350255,"name":"Alayna Bland","orcid":null,"position":4,"is_corresponding":false},{"id":1350256,"name":"Victoria Ehrman","orcid":null,"position":5,"is_corresponding":false},{"id":91662,"name":"A. Joachimiak","orcid":"0000-0003-2535-6209","position":6,"is_corresponding":false},{"id":1349948,"name":"Kenneth W. Olsen","orcid":"0000-0001-7116-6321","position":7,"is_corresponding":false},{"id":380826,"name":"Daniel P. Becker","orcid":"0000-0001-9392-0460","position":8,"is_corresponding":false},{"id":1349946,"name":"Emma H. Kelley","orcid":"0000-0002-3638-7628","position":0,"is_corresponding":true}],"reference_count":70,"raw_metadata":null,"created_at":"2026-07-19T02:10:08.435215Z","pmid":"39055043","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":[]}