{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:54:13Z","timestamp":1787032453515,"version":"build-2736575974"},"posted":{"date-parts":[[2026,5,5]]},"group-title":"Biochemistry","reference-count":62,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T00:00:00Z","timestamp":1777939200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R35GM118035"],"award-info":[{"award-number":["R35GM118035"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM143054"],"award-info":[{"award-number":["R35GM143054"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["F31DA060484"],"award-info":[{"award-number":["F31DA060484"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["Hanna H. Gray Fellowship"],"award-info":[{"award-number":["Hanna H. Gray Fellowship"]}],"id":[{"id":"https:\/\/ror.org\/006w34k90","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000861","name":"Burroughs Wellcome Fund","doi-asserted-by":"crossref","award":["Postdoctoral Enrichment Grant"],"award-info":[{"award-number":["Postdoctoral Enrichment Grant"]}],"id":[{"id":"10.13039\/100000861","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced by biosynthetic enzymes that modify genetically encoded precursor peptide backbones and side chains. Genome mining and bioinformatics analyses targeting the multinuclear nonheme iron oxidative (MNIO) enzyme family led to the identification of a\n                  <jats:italic>Streptomyces thermodiastaticus<\/jats:italic>\n                  JCM 4840 RiPP biosynthetic gene cluster, the\n                  <jats:italic>std<\/jats:italic>\n                  cluster, which includes multiple biosynthetic enzymes and a precursor peptide containing a conserved SNKEWQE motif. Using in vitro approaches, we elucidated the modifications installed by the\n                  <jats:italic>std<\/jats:italic>\n                  biosynthetic enzymes. First, a YcaO-TfuA pair thioamidates the backbone of asparagine. Next, a peptidase S8\/S53 domain fused to a NodU-like carbamoyltransferase that both carbamoylates the \u03b5-amino group of lysine to produce the non-proteinogenic amino acid homocitrulline and cleaves the C-terminal EWQE motif. Finally, a partner protein-MNIO pair bis-hydroxylates the \u03b2- and \u03b3-carbon positions of the installed homocitrulline. The formation of homocitrulline and its subsequent modification are unprecedented in RiPP biosynthesis. Moreover, these findings expand the substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyl transferases in RiPP biosynthesis.\n                <\/jats:p>","DOI":"10.64898\/2026.05.01.722236","type":"posted-content","created":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T20:55:13Z","timestamp":1778014513000},"source":"Crossref","is-referenced-by-count":5,"title":["Bis-hydroxylation of Homocitrulline Catalyzed by a Multinuclear Nonheme Iron-Dependent Oxidative Enzyme during RiPP Biosynthesis"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1950-0331","authenticated-orcid":false,"given":"Dayna P.","family":"Hebron","sequence":"first","affiliation":[{"name":"Departments of Molecular Biosciences and of Chemistry, Northwestern University, Evanston, Illinois 60208, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5121-8881","authenticated-orcid":false,"given":"Tucker J.","family":"Shriver","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3740-9999","authenticated-orcid":false,"given":"Joshua J.","family":"Ziarek","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8472-4134","authenticated-orcid":false,"given":"Amy C.","family":"Rosenzweig","sequence":"additional","affiliation":[{"name":"Departments of Molecular Biosciences and of Chemistry, Northwestern University, Evanston, Illinois 60208, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026051022250895000_2026.05.01.722236v1.1","doi-asserted-by":"publisher","DOI":"10.1039\/D0NP00027B"},{"key":"2026051022250895000_2026.05.01.722236v1.2","doi-asserted-by":"publisher","DOI":"10.1039\/c2np20085f"},{"key":"2026051022250895000_2026.05.01.722236v1.3","doi-asserted-by":"crossref","first-page":"1838","DOI":"10.1016\/j.csbj.2020.06.032","article-title":"Genome Mining Strategies for Ribosomally Synthesised and Post-Translationally Modified Peptides","volume":"18","year":"2020","journal-title":"Comput Struct Biotechnol J"},{"key":"2026051022250895000_2026.05.01.722236v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2017.02.005"},{"key":"2026051022250895000_2026.05.01.722236v1.5","doi-asserted-by":"crossref","first-page":"16377","DOI":"10.1039\/D5SC90158H","article-title":"A Focus on Unexpected Surprises in RiPP Natural Product Biosynthesis","volume":"16","year":"2025","journal-title":"Chem Sci"},{"key":"2026051022250895000_2026.05.01.722236v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2024.102467"},{"key":"2026051022250895000_2026.05.01.722236v1.7","first-page":"89","article-title":"Genome Mining for Natural Products Made by Multinuclear Iron-Dependent Oxidation Enzymes (MNIOs)","volume":"717","year":"2025","journal-title":"Methods Enzymol"},{"key":"2026051022250895000_2026.05.01.722236v1.8","doi-asserted-by":"publisher","DOI":"10.1126\/science.aap9437"},{"key":"2026051022250895000_2026.05.01.722236v1.9","doi-asserted-by":"publisher","DOI":"10.1126\/science.aau6232"},{"key":"2026051022250895000_2026.05.01.722236v1.10","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1021\/acscentsci.3c00160","article-title":"Macrocyclization and Backbone Rearrangement During RiPP Biosynthesis by a SAM-Dependent Domain-of-Unknown-Function 692","volume":"9","year":"2023","journal-title":"ACS Cent Sci"},{"key":"2026051022250895000_2026.05.01.722236v1.11","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1021\/acschembio.5c00987","article-title":"Biosynthesis of Peptidic Thiooxazole Metallophores Installed by Multinuclear Nonheme Iron Enzymes","volume":"21","year":"2026","journal-title":"ACS Chem Biol"},{"key":"2026051022250895000_2026.05.01.722236v1.12","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2408304121"},{"key":"2026051022250895000_2026.05.01.722236v1.13","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2408092121"},{"key":"2026051022250895000_2026.05.01.722236v1.14","doi-asserted-by":"crossref","first-page":"2462","DOI":"10.1021\/acschembio.5c00507","article-title":"Phylogenomic Identification of a Highly Conserved Copper-Binding RiPP Biosynthetic Gene Cluster in Marine Microbulbifer Bacteria","volume":"20","year":"2025","journal-title":"ACS Chem Biol"},{"key":"2026051022250895000_2026.05.01.722236v1.15","first-page":"e2532672123","article-title":"Co-Opting the Bacterial Lipoprotein Pathway in the Biosynthesis of a Lipidated Macrocyclic Peptide","volume":"123","year":"2025","journal-title":"Proc Natl Acad Sci"},{"key":"2026051022250895000_2026.05.01.722236v1.16","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.3c11740"},{"key":"2026051022250895000_2026.05.01.722236v1.17","doi-asserted-by":"crossref","first-page":"1022","DOI":"10.1021\/acscentsci.4c00088","article-title":"Biosynthesis of Macrocyclic Peptides with C-Terminal beta-Amino-alpha-keto Acid Groups by Three Different Metalloenzymes","volume":"10","year":"2024","journal-title":"ACS Cent Sci"},{"key":"2026051022250895000_2026.05.01.722236v1.18","doi-asserted-by":"crossref","first-page":"3551","DOI":"10.1021\/jacs.5c19246","article-title":"Oxidative Peptide Backbone Cleavage by a HEXXH Enzyme during RiPP Biosynthesis","volume":"148","year":"2026","journal-title":"J Am Chem Soc"},{"key":"2026051022250895000_2026.05.01.722236v1.19","doi-asserted-by":"crossref","unstructured":"Cha, L. ; Qian, C. ; Padhi, C. ; Zhu, L. ; van der Donk, W. A. , Discovery and Biosynthesis of Nitrilobacillins by Post-translational Introduction of C-Terminal Nitrile Groups. bioRxiv 2026.","DOI":"10.64898\/2026.03.11.711119"},{"key":"2026051022250895000_2026.05.01.722236v1.20","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.5c06044"},{"key":"2026051022250895000_2026.05.01.722236v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2023.168018"},{"key":"2026051022250895000_2026.05.01.722236v1.22","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkr1044"},{"key":"2026051022250895000_2026.05.01.722236v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09065-5"},{"key":"2026051022250895000_2026.05.01.722236v1.24","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.1c05017"},{"key":"2026051022250895000_2026.05.01.722236v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41557-022-01022-y"},{"key":"2026051022250895000_2026.05.01.722236v1.26","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.8b10724"},{"key":"2026051022250895000_2026.05.01.722236v1.27","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1021\/acs.biochem.4c00661","article-title":"Cysimiditides: RiPPs with a Zn-Tetracysteine Motif and Aspartimidylation","volume":"64","year":"2025","journal-title":"Biochemistry"},{"key":"2026051022250895000_2026.05.01.722236v1.28","doi-asserted-by":"crossref","first-page":"2143","DOI":"10.1021\/acscentsci.5c01243","article-title":"A Versatile Enzymatic Pathway for Modification of Peptide C-Termini","volume":"11","year":"2025","journal-title":"ACS Cent Sci"},{"key":"2026051022250895000_2026.05.01.722236v1.29","doi-asserted-by":"crossref","first-page":"635389","DOI":"10.3389\/fmicb.2021.635389","article-title":"Global Genome Mining Reveals the Distribution of Diverse Thioamidated RiPP Biosynthesis Gene Clusters","volume":"12","year":"2021","journal-title":"Front Microbiol"},{"key":"2026051022250895000_2026.05.01.722236v1.30","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1094\/MPMI.1999.12.1.68","article-title":"Carbamoylation of Azorhizobial Nod Factors is Mediated by NodU","volume":"12","year":"1999","journal-title":"Mol Plant Microbe Interact"},{"key":"2026051022250895000_2026.05.01.722236v1.31","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.0c00109"},{"key":"2026051022250895000_2026.05.01.722236v1.32","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1021\/acschembio.8b01058","article-title":"Salinipeptins: Integrated Genomic and Chemical Approaches Reveal Unusual d-Amino Acid-Containing Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs) from a Great Salt Lake Streptomyces sp","volume":"14","year":"2019","journal-title":"ACS Chem Biol"},{"key":"2026051022250895000_2026.05.01.722236v1.33","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-33890-w"},{"key":"2026051022250895000_2026.05.01.722236v1.34","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2123566119"},{"key":"2026051022250895000_2026.05.01.722236v1.35","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-022-00620-2"},{"key":"2026051022250895000_2026.05.01.722236v1.36","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1722324115"},{"key":"2026051022250895000_2026.05.01.722236v1.37","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.2c00402"},{"key":"2026051022250895000_2026.05.01.722236v1.38","doi-asserted-by":"publisher","DOI":"10.1039\/d0sc06835g"},{"key":"2026051022250895000_2026.05.01.722236v1.39","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.6b00623"},{"key":"2026051022250895000_2026.05.01.722236v1.40","doi-asserted-by":"crossref","first-page":"6617","DOI":"10.1038\/s41467-022-34387-2","article-title":"Endowing Homodimeric Carbamoyltransferase GdmN with Iterative Functions Through Structural Characterization and Mechanistic Studies","volume":"13","year":"2022","journal-title":"Nat Commun"},{"key":"2026051022250895000_2026.05.01.722236v1.41","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-021-00771-0"},{"key":"2026051022250895000_2026.05.01.722236v1.42","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1021\/acschembio.8b01022","article-title":"Biosynthesis and Chemical Applications of Thioamides","volume":"14","year":"2019","journal-title":"ACS Chem Biol"},{"key":"2026051022250895000_2026.05.01.722236v1.43","doi-asserted-by":"publisher","DOI":"10.1038\/ja.2006.2"},{"key":"2026051022250895000_2026.05.01.722236v1.44","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.7b00676"},{"key":"2026051022250895000_2026.05.01.722236v1.45","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz192"},{"key":"2026051022250895000_2026.05.01.722236v1.46","doi-asserted-by":"crossref","first-page":"1002","DOI":"10.1021\/acs.jnatprod.0c01027","article-title":"Targeted Isolation of Saalfelduracin B-D from Amycolatopsis saalfeldensis Using LC-MS\/MS-Based Molecular Networking","volume":"84","year":"2021","journal-title":"J Nat Prod"},{"key":"2026051022250895000_2026.05.01.722236v1.47","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.8b04238"},{"key":"2026051022250895000_2026.05.01.722236v1.48","doi-asserted-by":"crossref","first-page":"16472","DOI":"10.1002\/anie.202102571","article-title":"Nocathioamides, Uncovered by a Tunable Metabologenomic Approach, Define a Novel Class of Chimeric Lanthipeptides","volume":"60","year":"2021","journal-title":"Angew Chem Int Ed Engl"},{"key":"2026051022250895000_2026.05.01.722236v1.49","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201108896"},{"key":"2026051022250895000_2026.05.01.722236v1.50","doi-asserted-by":"crossref","first-page":"1198","DOI":"10.1021\/acschembio.5b01001","article-title":"The O-Carbamoyl-Transferase Alb15 Is Responsible for the Modification of Albicidin","volume":"11","year":"2016","journal-title":"ACS Chem Biol"},{"key":"2026051022250895000_2026.05.01.722236v1.51","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0403526101"},{"key":"2026051022250895000_2026.05.01.722236v1.52","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201412190"},{"key":"2026051022250895000_2026.05.01.722236v1.53","doi-asserted-by":"crossref","first-page":"7407","DOI":"10.1002\/anie.201612640","article-title":"Structure and Biosynthesis of Crocagins: Polycyclic Posttranslationally Modified Ribosomal Peptides from Chondromyces crocatus","volume":"56","year":"2017","journal-title":"Angew Chem Int Ed Engl"},{"key":"2026051022250895000_2026.05.01.722236v1.54","doi-asserted-by":"crossref","first-page":"560","DOI":"10.1038\/s41557-023-01153-w","article-title":"Unusual Peptide-Binding Proteins Guide Pyrroloindoline Alkaloid Formation in Crocagin Biosynthesis","volume":"15","year":"2023","journal-title":"Nat Chem"},{"key":"2026051022250895000_2026.05.01.722236v1.55","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1517096113"},{"key":"2026051022250895000_2026.05.01.722236v1.56","first-page":"1","article-title":"Protein Carbamylation: Chemistry, Pathophysiological Involvement, and Biomarkers","volume":"84","year":"2018","journal-title":"Adv Clin Chem"},{"key":"2026051022250895000_2026.05.01.722236v1.57","first-page":"271","article-title":"Separation of Ornithine and Lysine Activities of the Ornithine-Transcarbamylase-catalyzed Reaction","volume":"29","year":"1983","journal-title":"Enzyme"},{"key":"2026051022250895000_2026.05.01.722236v1.58","first-page":"26","article-title":"Further Evidence for a Separate Enzymic Entity for the Synthesis Oof Homocitrulline, Distinct from the Regular Ornithine Transcarbamylase","volume":"32","year":"1984","journal-title":"Enzyme"},{"key":"2026051022250895000_2026.05.01.722236v1.59","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.0c00685"},{"key":"2026051022250895000_2026.05.01.722236v1.60","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.2340"},{"key":"2026051022250895000_2026.05.01.722236v1.61","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.437947"},{"key":"2026051022250895000_2026.05.01.722236v1.62","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-024-01736-9"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.01.722236","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T05:25:20Z","timestamp":1778477120000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.01.722236"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,5]]},"references-count":62,"URL":"https:\/\/doi.org\/10.64898\/2026.05.01.722236","relation":{},"subject":[],"published":{"date-parts":[[2026,5,5]]},"subtype":"preprint"}}