Bis-hydroxylation of Homocitrulline Catalyzed by a Multinuclear Nonheme Iron Oxidative Enzyme during RiPP Biosynthesis

biosynthetic enzymes. First, a YcaO-TfuA pair thioamidates the asparagine backbone. Next, a peptidase with an S8/S53 domain fused to a NodU-like carbamoyltransferase both carbamoylates the ε-amino group of lysine to produce the nonproteinogenic amino acid homocitrulline and cleaves the C-terminal EWQE motif. Finally, a partner protein-MNIO pair bis-hydroxylates the β- and γ-carbon positions of the installed homocitrulline to create dihydroxyhomocitrulline. The formation of homocitrulline and dihydroxyhomocitrulline is unprecedented in RiPP biosynthesis. Moreover, these findings expand the known substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyltransferases in these pathways.