Sinefungin Biosynthesis Employs a B ₁₂ -Dependent Radical <i>S</i> -Adenosyl- <scp>l</scp> -methionine Enzyme Catalyzing C-Adenosylation of Arginine

Sinefungin is a nucleoside natural product isolated from several strains of Streptomyces . The chemical structure of sinefungin is characterized by an unusual C–C bond linkage between ornithine and adenosyl fragments. Previous studies suggested that pyridoxal 5′-phosphate (PLP)-dependent enzyme catalysis is involved in the key C–C bond-forming step. Here, we show that instead of PLP catalysis, sinefungin biosynthesis involves a B 12 -dependent radical S -adenosyl- l -methionine (SAM) enzyme, SnfB, that catalyzes the C-adenosylation of arginine. The amidino group in the resulting SnfB product is then hydrolyzed by SnfC to produce sinefungin via a cryptic phosphorylation step catalyzed by SnfH. This study highlights the catalytic versatility of the B 12 -dependent radical SAM enzyme family and unveils an unconventional assembly pathway for amino acid-nucleoside conjugates in nature.