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Among these compounds, phenazine dimers have attracted considerable attention owing to their intricate molecular architectures and potent antibacterial and anticancer properties. Although the biosynthesis of the core phenazine scaffold is well characterized, the enzymatic postmodifications, especially those governing dimerization processes that yield structurally varied diphenazines, remain poorly understood. In this study, we elucidate the complete biosynthesis of diverse dimeric and aminated phenazines. Following core assembly, the flavoprotein DapS initiates postmodification by catalyzing the C6-selective decarboxylative hydroxylation of phenazine-1,6-dicarboxylic acid (PDC). We further uncover Dap5, a nuclear transport factor 2 (NTF2)-like protein, as a multifunctional enzyme that catalyzes hydroxylation, dimerization, and amination of phenazines. Strikingly, Dap5 operates independently of flavin or metal cofactors, instead activating oxygen by exploiting the redox cycling of its phenazine substrate within the enzyme\u2013substrate complex. Structural and docking analyses based on a modeled protein structure suggest that these reactions likely share a common predicted active-site cavity but employ distinct residue networks for substrate binding and catalysis. This study solves a long-standing puzzle in the biosynthesis of dimeric phenazines and demonstrates a unique substrate-assisted radical strategy, offering new insights into the evolution of multifunctional enzymes.<\/jats:p>","DOI":"10.1021\/jacsau.6c01252","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T16:28:43Z","timestamp":1788452923000},"page":"5455-5464","source":"Crossref","is-referenced-by-count":0,"title":["Biosynthesis of Complex\nPhenazines Involves Dimerization\nand Amination Catalyzed by a Versatile Nuclear Transport Factor 2-like\nSuperfamily Protein"],"prefix":"10.1021","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-5900-2459","authenticated-orcid":true,"given":"Jun-Li","family":"Dong","sequence":"first","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Dan","sequence":"additional","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Xu","sequence":"additional","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bei-Bei","family":"He","sequence":"additional","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Huang","sequence":"additional","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing-Jing","family":"Xu","sequence":"additional","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing-Bo","family":"Liang","sequence":"additional","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9310-5666","authenticated-orcid":true,"given":"Yi-Qing","family":"Li","sequence":"additional","affiliation":[{"name":"Yunnan University , , ,","place":["Kunming, People\u2019s Republic of China, 650091"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2780-0978","authenticated-orcid":true,"given":"Li","family":"Han","sequence":"additional","affiliation":[{"name":"Northeastern University , , ,","place":["Shenyang, People\u2019s Republic of China, 110819"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1561-8108","authenticated-orcid":true,"given":"Xue-Shi","family":"Huang","sequence":"additional","affiliation":[{"name":"Northeastern University , , ,","place":["Shenyang, People\u2019s Republic of China, 110819"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1392-6040","authenticated-orcid":true,"given":"Jing","family":"He","sequence":"additional","affiliation":[{"name":"Huazhong Agricultural University , , No. 1 Shizishan Street , ,","place":["Wuhan, People\u2019s Republic of China, 430070"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,2]]},"reference":[{"issue":"3","key":"2026092808091823700_cit1","doi-asserted-by":"publisher","first-page":"1663","DOI":"10.1021\/cr020473j","article-title":"Phenazine natural products: biosynthesis, synthetic\nanalogues, and biological activity","volume":"104","author":"Laursen","year":"2004","journal-title":"Chem. Rev."},{"issue":"22","key":"2026092808091823700_cit2","doi-asserted-by":"publisher","first-page":"6149","DOI":"10.1016\/j.bmc.2017.01.002","article-title":"Recent developments in the isolation,\nbiological function, biosynthesis, and synthesis of phenazine natural\nproducts","volume":"25","author":"Guttenberger","year":"2017","journal-title":"Bioorg. Med. Chem."},{"issue":"29C","key":"2026092808091823700_cit3","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1016\/j.sbi.2014.08.013","article-title":"The structural biology\nof phenazine biosynthesis","volume":"29","author":"Blankenfeldt","year":"2014","journal-title":"Curr. Opin. Struct.\nBiol."},{"issue":"10","key":"2026092808091823700_cit4a","first-page":"1659","article-title":"Endophytic Fungus Nigrospora oryzae from a medicinal\nplant Coccinia grandis, a high yielding new source of phenazine-1-carboxamide","volume":"10","author":"Thanabalasingam","year":"2015","journal-title":"Nat. Prod. Commun."},{"issue":"7","key":"2026092808091823700_cit4b","doi-asserted-by":"publisher","first-page":"445","DOI":"10.1038\/ja.2015.14","article-title":"Identification of novel endophenaside\nantibiotics produced by Kitasatospora sp. MBT66","volume":"68","author":"Wu","year":"2015","journal-title":"J. Antibiot. (Tokyo)"},{"issue":"4","key":"2026092808091823700_cit5","doi-asserted-by":"publisher","first-page":"331","DOI":"10.1038\/s41589-019-0246-1","article-title":"Dual phenazine\ngene clusters enable\ndiversification during biosynthesis","volume":"15","author":"Shi","year":"2019","journal-title":"Nat. Chem.\nBiol."},{"issue":"6","key":"2026092808091823700_cit6","doi-asserted-by":"publisher","first-page":"1686","DOI":"10.1021\/acs.jnatprod.9b00289","article-title":"Baraphenazines\nA-G, divergent fused phenazine-based metabolites from a Himalayan\nStreptomyces","volume":"82","author":"Wang","year":"2019","journal-title":"J. Nat. Prod."},{"issue":"12","key":"2026092808091823700_cit7","doi-asserted-by":"publisher","first-page":"1999","DOI":"10.1021\/np100400t","article-title":"Izumiphenazines\nA-C: isolation and\nstructure elucidation of phenazine derivatives from Streptomyces sp.\nIFM 11204","volume":"73","author":"Abdelfattah","year":"2010","journal-title":"J. Nat. Prod."},{"issue":"4","key":"2026092808091823700_cit8","doi-asserted-by":"publisher","first-page":"857","DOI":"10.1021\/acs.jnatprod.1c00985","article-title":"Isolation,\nStructure Determination, and Semisynthesis of Diphenazine Compounds\nfrom a Deep-Sea-Derived Strain of the Fungus Cystobasidium laryngis\nand Their Biological Activities","volume":"85","author":"Lee","year":"2022","journal-title":"J. Nat. 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(Tokyo)"},{"issue":"19","key":"2026092808091823700_cit11","doi-asserted-by":"publisher","first-page":"4771","DOI":"10.3390\/molecules29194771","article-title":"Recent Advances in Phenazine Natural\nProducts: Chemical Structures and Biological Activities","volume":"29","author":"Huang","year":"2024","journal-title":"Molecules"},{"issue":"4","key":"2026092808091823700_cit12","doi-asserted-by":"publisher","first-page":"1168","DOI":"10.1111\/1751-7915.13909","article-title":"Characterization\nof the diastaphenazine\/izumiphenazine\nC biosynthetic gene cluster from plant endophyte Streptomyces diastaticus\nW2","volume":"15","author":"Dong","year":"2022","journal-title":"Microb. Biotechnol."},{"issue":"7","key":"2026092808091823700_cit13","doi-asserted-by":"publisher","first-page":"1801","DOI":"10.1021\/acs.jnatprod.3c00306","article-title":"An ECD and NMR\/DP4+\nComputational Pipeline for Structure Revision and Elucidation of Diphenazine-Based\nNatural Products","volume":"86","author":"Zhuang","year":"2023","journal-title":"J. Nat. 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Mol. Biol."},{"issue":"44","key":"2026092808091823700_cit17","doi-asserted-by":"publisher","first-page":"18490","DOI":"10.1039\/D4SC05590J","article-title":"Three-dimensional structural alignment based discovery\nand molecular basis of AtoB, catalyzing linear tetracyclic formation","volume":"15","author":"Ma","year":"2024","journal-title":"Chem. Sci."},{"issue":"10","key":"2026092808091823700_cit18","doi-asserted-by":"publisher","first-page":"731","DOI":"10.1038\/ja.2016.88","article-title":"Crystal structure of SgcJ, an NTF2-like\nsuperfamily\nprotein involved in biosynthesis of the nine-membered enediyne antitumor\nantibiotic C-1027","volume":"69","author":"Huang","year":"2016","journal-title":"J. Antibiot."},{"key":"2026092808091823700_cit19","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1002\/mrc.1270190203","article-title":"1H NMR spectra of substituted\nphenazines","volume":"19","author":"Ro\u0308mer","year":"1982","journal-title":"Magn.\nReson. Chem."},{"issue":"1","key":"2026092808091823700_cit20","doi-asserted-by":"publisher","first-page":"17","DOI":"10.11554\/antibioticsa.12.1_17","article-title":"1, 6-Di-hydroxyphenazine, an antibiotic produced by\nStreptomyces thioluteus","volume":"12","author":"Akabori","year":"1959","journal-title":"J. Antibiot. (Tokyo)"},{"issue":"19","key":"2026092808091823700_cit21","doi-asserted-by":"publisher","first-page":"5281","DOI":"10.1021\/bi702480t","article-title":"Crystal\nstructure of the pyocyanin biosynthetic protein\nPhzS","volume":"47","author":"Greenhagen","year":"2008","journal-title":"Biochemistry"},{"issue":"10","key":"2026092808091823700_cit22","doi-asserted-by":"publisher","first-page":"2495","DOI":"10.1021\/acs.orglett.6b01089","article-title":"Heterocyclic aromatic\nN-Oxidation in the biosynthesis of phenazine antibiotics from Lysobacter\nantibioticus","volume":"18","author":"Zhao","year":"2016","journal-title":"Org. Lett."},{"issue":"9","key":"2026092808091823700_cit23","doi-asserted-by":"publisher","first-page":"1911","DOI":"10.1038\/sj.emboj.7600201","article-title":"Structure\nof the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic\naldol condensation","volume":"23","author":"Sultana","year":"2004","journal-title":"EMBO J."},{"issue":"6","key":"2026092808091823700_cit24","doi-asserted-by":"publisher","first-page":"5391","DOI":"10.1021\/acscatal.9b01034","article-title":"Enzymatic Pyran\nFormation Involved in Xiamenmycin Biosynthesis","volume":"9","author":"He","year":"2019","journal-title":"ACS Catal."},{"issue":"7750","key":"2026092808091823700_cit25","doi-asserted-by":"publisher","first-page":"122","DOI":"10.1038\/s41586-019-1021-x","article-title":"Enzyme-catalysed [6 + 4] cycloadditions in the biosynthesis\nof natural\nproducts","volume":"568","author":"Zhang","year":"2019","journal-title":"Nature"},{"issue":"11","key":"2026092808091823700_cit26a","doi-asserted-by":"publisher","first-page":"1308","DOI":"10.7164\/antibiotics.37.1308","article-title":"Phenacein\u2013an angiotensin-converting\nenzyme inhibitor produced by a streptomycete. I. Taxonomy, fermentation\nand biological properties","volume":"37","author":"Bush","year":"1984","journal-title":"J. Antibiot. (Tokyo)"},{"issue":"11","key":"2026092808091823700_cit26b","doi-asserted-by":"publisher","first-page":"1313","DOI":"10.7164\/antibiotics.37.1313","article-title":"Phenacein - an angiotensin-converting\nenzyme inhibitor produced by a streptomycete. II. Isolation, structure\ndetermination and synthesis","volume":"37","author":"Liu","year":"1984","journal-title":"J. Antibiot."},{"issue":"Pt 3","key":"2026092808091823700_cit27","doi-asserted-by":"publisher","first-page":"156501","DOI":"10.1016\/j.scitotenv.2022.156501","article-title":"Investigating\nthe interaction between\nShewanella oneidensis and phenazine 1-carboxylic acid in the microbial\nelectrochemical processes","volume":"838","author":"Yu","year":"2022","journal-title":"Sci. Total Environ."},{"issue":"7","key":"2026092808091823700_cit28a","doi-asserted-by":"publisher","first-page":"755","DOI":"10.1007\/BF00471348","article-title":"Mechanism of the photochemical\naddition-substitution\nreactions of six-membered aza aromatic compounds in hydrogen-containing\nsolvents","volume":"10","author":"Castellano","year":"1974","journal-title":"Chem. Heterocycl. Compd."},{"issue":"13","key":"2026092808091823700_cit28b","doi-asserted-by":"publisher","first-page":"2816","DOI":"10.1021\/ol9009927","article-title":"Pyrene-dihydrophenazine\nbis (radical cation) in a singlet ground state","volume":"11","author":"Suzuki","year":"2009","journal-title":"Org. Lett."},{"issue":"16","key":"2026092808091823700_cit29","doi-asserted-by":"publisher","first-page":"6611","DOI":"10.1021\/bi00442a012","article-title":"Effects of distal\npoint-site mutations on the binding and catalysis of dihydrofolate\nreductase from Escherichia coli","volume":"28","author":"Adams","year":"1989","journal-title":"Biochemistry"},{"issue":"18","key":"2026092808091823700_cit30","doi-asserted-by":"publisher","first-page":"16246","DOI":"10.1074\/jbc.M111.230532","article-title":"Conformational\nchanges of NADPH-cytochrome P450 oxidoreductase\nare essential for catalysis and cofactor binding","volume":"286","author":"Xia","year":"2011","journal-title":"J. Biol. Chem."},{"issue":"52","key":"2026092808091823700_cit31","doi-asserted-by":"publisher","first-page":"27166","DOI":"10.1002\/ange.202109384","article-title":"Catalytic control of\nspiroketal formation in rubromycin polyketide biosynthesis","volume":"133","author":"Toplak","year":"2021","journal-title":"Angew. Chem."},{"issue":"7","key":"2026092808091823700_cit32","doi-asserted-by":"publisher","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular\nEvolutionary Genetics\nAnalysis Version 7.0 for Bigger Datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol.\nBiol. Evol."},{"issue":"7873","key":"2026092808091823700_cit33","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"issue":"6","key":"2026092808091823700_cit34","doi-asserted-by":"publisher","first-page":"679","DOI":"10.1038\/s41592-022-01488-1","article-title":"ColabFold: making protein folding\naccessible to all","volume":"19","author":"Mirdita","year":"2022","journal-title":"Nat. Methods"}],"container-title":["JACS Au"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacsau.6c01252","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jaaucr\/article-pdf\/6\/9\/5455\/68209072\/jacsau.6c01252.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jaaucr\/article-pdf\/6\/9\/5455\/68209072\/jacsau.6c01252.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T12:30:31Z","timestamp":1790598631000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jaaucr\/article\/6\/9\/5455\/5405821\/Biosynthesis-of-Complex-Phenazines-Involves"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,2]]},"references-count":37,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9,2]]},"published-print":{"date-parts":[[2026,9,28]]}},"URL":"https:\/\/doi.org\/10.1021\/jacsau.6c01252","relation":{},"ISSN":["2691-3704"],"issn-type":[{"value":"2691-3704","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,2]]}}}