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Here, we disclose detailed mechanistic insights by characterizing all intermediates in the sequential steps of decarboxylation, desaturation, and epoxidation with synthetic flavins and the flavin\u2010dependent oxidoreductase EpxF. A high\u2010resolution crystal structure of EpxF revealed the architecture of the active site and enabled the identification of key catalytic residues. Exploratory docking based on this structure served as a qualitative tool to guide mutagenesis and rationalize substrate recognition. NMR studies with a\n                    <jats:sup>13<\/jats:sup>\n                    C\u2010labeled epoxomicin precursor and structure\u2010based EpxF variants further supported the proposed mechanism. Our integrated approach revealed similarities between synthetic and natural flavin catalysts and offers avenues for developing sustainable biomimetic reactions.\n                  <\/jats:p>","DOI":"10.1002\/anie.202512568","type":"journal-article","created":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T12:00:17Z","timestamp":1760443217000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Studies of \u03b1\u2032,\u03b2\u2032\u2010Epoxyketone Synthesis by Small\u2010Molecule Flavins and Flavoenzymes"],"prefix":"10.1002","volume":"64","author":[{"given":"Alexandra","family":"Walter","sequence":"first","affiliation":[{"name":"Technical University of Munich (TUM) School of Natural Sciences and Catalysis Research Center (CRC)  Lichtenbergstr. 4 85747 Garching Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2359-3019","authenticated-orcid":false,"given":"Wolfgang","family":"Kuttenlochner","sequence":"additional","affiliation":[{"name":"Technical University of Munich (TUM) School of Natural Sciences and Center for Protein Assemblies (CPA)  Ernst\u2010Otto\u2010Fischer\u2010Stra\u00dfe 8 85748 Garching Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9832-8279","authenticated-orcid":false,"given":"Wolfgang","family":"Eisenreich","sequence":"additional","affiliation":[{"name":"Technical University of Munich (TUM) School of Natural Sciences and Bavarian NMR Center (BNMRZ), Structural Membrane Biochemistry  Lichtenbergstr. 4 85747 Garching Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengyang","family":"Yao","sequence":"additional","affiliation":[{"name":"Technical University of Munich (TUM) School of Natural Sciences and Center for Protein Assemblies (CPA)  Ernst\u2010Otto\u2010Fischer\u2010Stra\u00dfe 8 85748 Garching Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1660-340X","authenticated-orcid":false,"given":"Michael","family":"Groll","sequence":"additional","affiliation":[{"name":"Technical University of Munich (TUM) School of Natural Sciences and Center for Protein Assemblies (CPA)  Ernst\u2010Otto\u2010Fischer\u2010Stra\u00dfe 8 85748 Garching Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6747-3035","authenticated-orcid":false,"given":"Golo","family":"Storch","sequence":"additional","affiliation":[{"name":"Technical University of Munich (TUM) School of Natural Sciences and Catalysis Research Center (CRC)  Lichtenbergstr. 4 85747 Garching Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,10,14]]},"reference":[{"key":"e_1_2_6_1_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201207900"},{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201201616"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.oprd.6b00374"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm801329v"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.8b01201"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1039\/c3np20126k"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.5b03688"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.18.10403"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.45.1746"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja993588m"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaf8993"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja3006463"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-894X(99)00376-5"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/cb400699p"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201500567"},{"key":"e_1_2_6_16_1","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.43.8"},{"key":"e_1_2_6_17_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.5b03833"},{"key":"e_1_2_6_18_1","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.50.261"},{"key":"e_1_2_6_19_1","first-page":"585","volume-title":"Methods Enzymol","author":"Kim K. 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