{"doi":"10.1101/2025.11.17.688841","title":"Structural Basis for Iterative Methylation by a Cobalamin-dependent Radical S-Adenosylmethionine Enzyme in Cystobactamids Biosynthesis","abstract":"Abstract Cystobactamids are non-ribosomal peptide natural products that function as DNA gyrase inhibitors, exhibiting significant antibacterial activity. They are isolated from Cystobacter sp. Cbv34 and contain various alkoxy groups on para-aminobenzoic acid moieties, which are believed to play a crucial role in antibacterial functions. The alkoxy groups are generated by iterative methylations on a methoxy group by the cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzyme CysS. CysS catalyzes up to three methylations to give ethoxy, isopropoxy, sec-butoxy, and tert-butoxy groups. For each methylation, CysS uses a ping-pong mechanism in which two molecules of SAM are consumed. One SAM is used to methylate cob(I)alamin, while another generates a 5′-deoxyadenosyl 5′-radical to initiate substrate methylation. However, little is known about how the enzyme promotes both Cbl methylation and iterative substrate methylation, which occur by polar S N 2 and radical processes, respectively. Here, we report three X-ray crystal structures of a homolog of CysS from Corallococcus sp. CA054B . Two were determined in the presence of methoxy- and ethoxy-containing substrates, showing how CysS accommodates substrates and products during iterative methylation. The third structure, determined in the absence of a substrate, exhibits structural changes that reorient the SAM’s conformation to allow for the methylation of cob(I)alamin.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":581612,"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":0.9541,"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":649878,"name":"Bo Wang","orcid":"0000-0002-0381-3686","position":1,"is_corresponding":false},{"id":1230039,"name":"Ravi K. Maurya","orcid":null,"position":2,"is_corresponding":false},{"id":649879,"name":"Squire J. Booker","orcid":"0000-0002-7211-5937","position":3,"is_corresponding":false},{"id":1230038,"name":"Jinhe Cui","orcid":null,"position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":null,"created_at":"2026-07-19T02:58:51.328454Z","pmid":"41332741","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":[]}