{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Padhi C"],"funding":["NCRR NIH HHS","NIGMS NIH HHS"],"pagination":["23781-23796"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC12257519"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["147(27)"],"pubmed_abstract":["Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a growing class of natural products. Multinuclear nonheme iron-dependent oxidative enzymes (MNIOs, previously DUF692) are involved in a range of unprecedented biochemical reactions. Over 13,500 putative MNIO-encoding biosynthetic gene clusters (BGCs) have been identified by sequence similarity networks. In this study, we investigated a set of precursor peptides containing a conserved FHAFRF motif in MNIO-encoding BGCs. These BGCs contain genes encoding an MNIO, a RiPP recognition element-containing protein, an arginase, a hydroxylase, and a vitamin B12-dependent radical SAM enzyme (B12-rSAM). Using heterologous reconstitution of a representative BGC from <i>Peribacillus simplex</i> (<i>pbs</i> cluster) in <i>E. coli</i>, we demonstrated that the MNIO in conjunction with the partner protein catalyzes <i>ortho</i>-hydroxylation of each of the phenylalanine residues in the conserved FRF motif, the arginase forms an ornithine from the arginine, the ornithine residue is hydroxylated, and the B12-rSAM cross-links the <i>ortho</i>-Tyr side chains by a C-C linkage forming a macrocycle. A protease matures the RiPP to its final form. The elucidated structure shares close similarity to biphenomycins, a class of peptide antibiotics for which the biosynthetic pathway has not been characterized. Substrate scope studies suggest some tolerance of the MNIO and the B12-rSAM enzymes. This study expands the diverse array of posttranslational modifications catalyzed by MNIOs and B12-rSAM enzymes, deorphanizes biphenomycin biosynthesis, and provides a platform for the production of analogs from orthologous BGCs."],"journal":["Journal of the American Chemical Society"],"pubmed_title":["Biosynthesis of Biphenomycin-like Macrocyclic Peptides by Formation and Cross-Linking of &lt;i&gt;Ortho&lt;/i&gt;-Tyrosines."],"pmcid":["PMC12257519"],"funding_grant_id":["S10 RR027109","R37 GM058822"],"pubmed_authors":["Moreira R","Huang C","Cryle MJ","Zhu L","Chen JY","van der Donk WA","Padhi C","Challis GL"],"additional_accession":[]},"is_claimable":false,"name":"Biosynthesis of Biphenomycin-like Macrocyclic Peptides by Formation and Cross-Linking of &lt;i&gt;Ortho&lt;/i&gt;-Tyrosines.","description":"Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a growing class of natural products. Multinuclear nonheme iron-dependent oxidative enzymes (MNIOs, previously DUF692) are involved in a range of unprecedented biochemical reactions. Over 13,500 putative MNIO-encoding biosynthetic gene clusters (BGCs) have been identified by sequence similarity networks. In this study, we investigated a set of precursor peptides containing a conserved FHAFRF motif in MNIO-encoding BGCs. These BGCs contain genes encoding an MNIO, a RiPP recognition element-containing protein, an arginase, a hydroxylase, and a vitamin B12-dependent radical SAM enzyme (B12-rSAM). Using heterologous reconstitution of a representative BGC from <i>Peribacillus simplex</i> (<i>pbs</i> cluster) in <i>E. coli</i>, we demonstrated that the MNIO in conjunction with the partner protein catalyzes <i>ortho</i>-hydroxylation of each of the phenylalanine residues in the conserved FRF motif, the arginase forms an ornithine from the arginine, the ornithine residue is hydroxylated, and the B12-rSAM cross-links the <i>ortho</i>-Tyr side chains by a C-C linkage forming a macrocycle. A protease matures the RiPP to its final form. The elucidated structure shares close similarity to biphenomycins, a class of peptide antibiotics for which the biosynthetic pathway has not been characterized. Substrate scope studies suggest some tolerance of the MNIO and the B12-rSAM enzymes. This study expands the diverse array of posttranslational modifications catalyzed by MNIOs and B12-rSAM enzymes, deorphanizes biphenomycin biosynthesis, and provides a platform for the production of analogs from orthologous BGCs.","dates":{"release":"2025-01-01T00:00:00Z","publication":"2025 Jul","modification":"2026-06-02T02:42:13.111Z","creation":"2026-04-13T03:12:24.061Z"},"accession":"S-EPMC12257519","cross_references":{"pubmed":["40568902"],"doi":["10.1021/jacs.5c06044"]}}