Abstract
Peptide natural products are important molecules for the development of efficient drugs for human health applications. The biphenomycins are bacterial macrocyclic peptides characterized by unique ortho-tyrosine (oTyr) residues connected by biaryl linkages. Biphenomycins possess potent antibacterial activity against Gram-positive pathogens at low doses with no eukaryotic toxicity. Despite their initial discovery in 1967, their biosynthetic pathway has remained elusive. Within this work, we identified the ribosomal biosynthetic origin of biphenomycins and elucidated all enzymatic maturation steps by in-depth functional characterization in vivo and in vitro. Key steps include selective ortho-hydroxylation events at two phenyl alanine residues catalyzed by a bifunctional multinuclear nonheme iron-dependent oxidase yielding the oTyr functionalities, biaryl cross coupling by a B12-dependent radical SAM enzyme, amino acid side-chain modifications by a highly regioselective arginase and by dedicated hydroxylases, as well as a stepwise proteolytic processing by a TldD-type but self-sufficient protease. These findings clarify the molecular basis of biphenomycin assembly, reveal unprecedented enzymatic dual functions, and provide the foundation for the targeted discovery of novel biphenomycins and for the development of bioengineering strategies to enhance yields and develop antibiotics with further increased potency, addressing the urgent need for new antimicrobial agents.