biostudies-literaturePadhi CNCRR NIH HHSNIGMS NIH HHS23781-23796https://www.ebi.ac.uk/biostudies/studies/S-EPMC12257519biostudies-literatureUnknown147(27)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 of the American Chemical SocietyBiosynthesis of Biphenomycin-like Macrocyclic Peptides by Formation and Cross-Linking of &lt;i&gt;Ortho&lt;/i&gt;-Tyrosines.PMC12257519S10 RR027109R37 GM058822Moreira RHuang CCryle MJZhu LChen JYvan der Donk WAPadhi CChallis GLfalseBiosynthesis of Biphenomycin-like Macrocyclic Peptides by Formation and Cross-Linking of &lt;i&gt;Ortho&lt;/i&gt;-Tyrosines.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.2025-01-01T00:00:00Z2025 Jul2026-06-02T02:42:13.111Z2026-04-13T03:12:24.061ZS-EPMC122575194056890210.1021/jacs.5c06044