{"doi":"10.1039/d5sc04563k","title":"Insights into the initial steps of the thiamin pyrimidine synthase (ThiC)-catalyzed reaction through EPR spectroscopic characterization of radical intermediates","abstract":"Thiamin pyrimidine synthase (ThiC) is a noncanonical radical SAM enzyme that catalyzes the complex radical rearrangement of aminoimidazole ribonucleotide (AIR) to hydroxymethylpyrimidine phosphate (HMP-P) as part of the thiamin biosynthetic pathway in bacteria and plants. In this work, we investigate the mechanism of ThiC using advanced electron paramagnetic resonance (EPR) techniques. Freeze-quenching a reaction of ThiC revealed the accumulation of a new radical species. By employing electron nuclear double resonance (ENDOR) spectroscopy with various substrate isotopologues, we determined the hyperfine parameters of several nuclei, allowing us to propose a structure for this intermediate. The accumulated species was characterized as a dihydro-aminoimidazole centered radical attached to two ribose derived fragments. This radical is sensitive to perturbations in the enzyme H-bonding network. In addition, mutagenesis of active site residues results in the accumulation of two distinct intermediates, including a C5' ribonucleotide centered radical and a ribose C2' radical fragment. Identification of these early radical intermediates provides insights into the initial steps of the ThiC mechanism. The ThiC-catalyzed reaction involves a 20-step radical cascade and is the most complex rearrangement found in biosynthesis. This study highlights the pivotal role that EPR can play in elucidating the mechanism of highly complicated enzyme-catalyzed reactions.","journal":"Chemical Science","year":2025,"id":583864,"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":0.9607,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1229352,"name":"Vishav Sharma","orcid":"0000-0002-2280-0549","position":1,"is_corresponding":false},{"id":1103457,"name":"Dmytro Fedoseyenko","orcid":"0000-0001-7776-8278","position":2,"is_corresponding":false},{"id":1440763,"name":"Yumeng Cao","orcid":"0000-0001-6049-7299","position":3,"is_corresponding":false},{"id":334946,"name":"Guodong Rao","orcid":"0000-0001-8043-3436","position":4,"is_corresponding":false},{"id":389027,"name":"Dean J. Tantillo","orcid":"0000-0002-2992-8844","position":5,"is_corresponding":false},{"id":793781,"name":"Tadhg P. Begley","orcid":"0000-0001-5134-2623","position":6,"is_corresponding":false},{"id":334945,"name":"R. David Britt","orcid":"0000-0003-0889-8436","position":7,"is_corresponding":false},{"id":488912,"name":"Melissa M. Bollmeyer","orcid":"0000-0002-8979-0584","position":0,"is_corresponding":true}],"reference_count":62,"raw_metadata":null,"created_at":"2026-07-19T02:59:07.970345Z","pmid":"41321715","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":[]}