{"doi":"10.1101/2025.10.30.685615","title":"Oxidative Peptide Backbone Cleavage by a HEXXH Enzyme During RiPP Biosynthesis","abstract":"Abstract Ribosomally synthesized and post-translationally modified peptides (RiPPs) rely on a diverse array of enzymes to tailor peptide backbones and side chains. In this study, we characterized enzymes from two different biosynthetic gene clusters (BGCs) from Pseudomonas strains ( pfl and pos ) that catalyze new transformations in RiPP biosynthesis. Two α-ketoglutarate-dependent HEXXH enzymes, PflC and PosC, perform hydroxylation of multiple consecutive glutamine residues and selectively recognize a C-terminal ARMD tetrapeptide to trigger oxidative backbone cleavage that generates an amide terminus. Mutational analysis pinpoints the first position of this motif as a critical determinant. Notably, PflC displays proteolytic activity in the absence of the leader peptide, indicating that leader peptide–enzyme interactions modulate the observed reaction selectivity. The biosynthetic gene clusters also encode a unique MNIO-nitroreductase fusion enzyme that installs a rare Z- dehydrophenylalanine and hydroxylates an Asp residue. Collectively, this work expands both the catalytic repertoire and structural diversity accessible through bacterial RiPP biosynthesis.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":559417,"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":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.962,"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":1174175,"name":"Yue Yu","orcid":"0000-0001-8306-4648","position":1,"is_corresponding":false},{"id":469874,"name":"Lingyang Zhu","orcid":"0000-0002-6657-271X","position":2,"is_corresponding":false},{"id":1173433,"name":"Dinh Thanh Nguyen","orcid":"0000-0002-2101-6808","position":3,"is_corresponding":false},{"id":256350,"name":"Wilfred A. van der Donk","orcid":"0000-0002-5467-7071","position":4,"is_corresponding":false},{"id":1460643,"name":"Yao Ouyang","orcid":"0000-0002-8379-5843","position":0,"is_corresponding":true}],"reference_count":40,"raw_metadata":null,"created_at":"2026-07-19T02:55:34.849815Z","pmid":"41279720","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":[]}