{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T08:58:00Z","timestamp":1785401880392,"version":"3.56.0"},"posted":{"date-parts":[[2026,5,23]]},"group-title":"Biochemistry","reference-count":52,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T00:00:00Z","timestamp":1779494400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R35 GM133475"],"award-info":[{"award-number":["R35 GM133475"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R37 GM058822"],"award-info":[{"award-number":["R37 GM058822"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Sinefungin is a potent nucleoside antimetabolite of\n                  <jats:italic>S<\/jats:italic>\n                  -adenosylmethionine (SAM), yet its biosynthesis has remained unclear for decades. Here we detail the identification and characterization of the complete sinefungin biosynthetic gene cluster (BGC) from\n                  <jats:italic>Streptomyces incarnatus<\/jats:italic>\n                  NRRL 8089. In vitro and in vivo analyses demonstrate that the defining carbon\u2013carbon (C\u2013C) bond is formed not by the long-hypothesized PLP-dependent process, but by a vitamin B\n                  <jats:sub>12<\/jats:sub>\n                  -dependent radical SAM enzyme. Using isotope-labeled cofactors and substrates, we provide evidence that the adenosyl group of sinefungin atypically originates from adenosylcobalamin via a homolytic\n                  <jats:italic>S<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>H<\/jats:italic>\n                  <\/jats:sub>\n                  2 substitution, establishing a rare instance where adenosylcobalamin is enzymatically consumed during the reaction. Furthermore, the pathway utilizes a cryptic phosphorylation-dephosphorylation strategy to control intermediate processing and substrate recognition. We also characterize two peptide aminoacyl-tRNA ligases (PEARLs) that append alanines onto the nucleoside scaffold using tRNA-activated amino acids. The PEARLs act directly on small molecules rather than macromolecular substrates, with one PEARL capable of iterative elongation. Finally, we leverage these enzymes in a reduced multi-enzyme cascade to biosynthesize sinefungin. Together, these findings redefine radical-mediated C\u2013C bond formation and pearlin enzyme versatility, unlocking biocatalytic possibilities to produce amino acid-nucleoside conjugates.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"726688v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.21.726688","type":"posted-content","created":{"date-parts":[[2026,5,24]],"date-time":"2026-05-24T02:20:16Z","timestamp":1779589216000},"source":"Crossref","is-referenced-by-count":0,"title":["Unconventional biocatalytic strategies orchestrate synthesis of the nucleoside analog sinefungin"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4582-9170","authenticated-orcid":false,"given":"Chi-Fang","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics & BioTechnology Institute, University of Minnesota Twin Cities, St. Paul, MN 55108, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2290-9632","authenticated-orcid":false,"given":"Tianhui H.","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-0684-8832","authenticated-orcid":false,"given":"Songyi","family":"Xue","sequence":"additional","affiliation":[{"name":"Department of Chemistry & Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6657-271X","authenticated-orcid":false,"given":"Lingyang","family":"Zhu","sequence":"additional","affiliation":[{"name":"School of Chemical Sciences NMR Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5467-7071","authenticated-orcid":false,"given":"Wilfred A.","family":"van der Donk","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"},{"name":"Department of Chemistry & Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"},{"name":"Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0491-9618","authenticated-orcid":false,"given":"Michael F.","family":"Freeman","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics & BioTechnology Institute, University of Minnesota Twin Cities, St. Paul, MN 55108, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026052702000573000_2026.05.21.726688v1.1","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.26.463"},{"key":"2026052702000573000_2026.05.21.726688v1.2","doi-asserted-by":"publisher","DOI":"10.1039\/d2np00086e"},{"key":"2026052702000573000_2026.05.21.726688v1.3","doi-asserted-by":"publisher","DOI":"10.1152\/physrev.00047.2011"},{"key":"2026052702000573000_2026.05.21.726688v1.4","doi-asserted-by":"publisher","DOI":"10.1186\/1472-6807-5-19"},{"key":"2026052702000573000_2026.05.21.726688v1.5","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.5b00812"},{"key":"2026052702000573000_2026.05.21.726688v1.6","doi-asserted-by":"publisher","DOI":"10.7164\/antibiotics.26.466"},{"key":"2026052702000573000_2026.05.21.726688v1.7","doi-asserted-by":"publisher","DOI":"10.3390\/app15094942"},{"key":"2026052702000573000_2026.05.21.726688v1.8","doi-asserted-by":"publisher","DOI":"10.1021\/ja307060p"},{"key":"2026052702000573000_2026.05.21.726688v1.9","doi-asserted-by":"publisher","DOI":"10.1042\/bj3050133"},{"key":"2026052702000573000_2026.05.21.726688v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/0014-4894(80)90010-7"},{"issue":"3","key":"2026052702000573000_2026.05.21.726688v1.11","first-page":"255","article-title":"Evaluation of Sinefungin for the Treatment of Trypanosoma (Nannomonas) congolense Infections in Goats","volume":"37","year":"1986","journal-title":"Trop. 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