Biosynthesis of Biphenomycin-like Macrocyclic Peptides by Formation and Cross-Linking of <i>Ortho</i>-Tyrosines.

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 Peribacillus simplex (pbs cluster) in E. coli, we demonstrated that the MNIO in conjunction with the partner protein catalyzes ortho-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 ortho-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.

SUBMITTER: Padhi C 

PROVIDER: S-EPMC12257519 | biostudies-literature | 2025 Jul

REPOSITORIES: biostudies-literature