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

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced by biosynthetic enzymes that modify genetically encoded precursor peptide backbones and side chains. Genome mining and bioinformatics analyses targeting the multinuclear nonheme iron oxidative (MNIO) enzyme family led to the identification of a RiPP biosynthetic gene cluster from Streptomyces thermodiastaticus JCM 4840, the std cluster, which includes multiple biosynthetic enzymes and a precursor peptide containing a conserved SNKEWQE motif. Using in vitro approaches, we elucidated the modifications installed by the std 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 non-proteinogenic 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.