NocU is a cytochrome P450 oxygenase catalyzing N-hydroxylation of the indolic moiety during the maturation of the thiopeptide antibiotics nocathiacins

The ribosomally synthesized and post-translationally modified peptide (RiPP) natural products include the family of thiopeptide antibiotics, where nocathiacins (NOCs) and nosiheptide (NOS) are structurally related bicyclic members featuring an indolic moiety within the side ring system. Compared with NOS, NOCs bear additional functionalities that lead to the improvement of water solubility and bioavailability, a problem inherent to most of the thiopeptide antibiotics, and thus hold potential for clinical use in anti-infective agent development. The process through which post-translational modifications (PTMs) occur to afford these functionalities remains unclear. In this study, an engineered NOS-producing strain is applied to study the function of NocU, a cytochrome P450 oxygenase unique during the PTMs in NOC biosynthesis. Benefiting from the isolation and structure characterization of nosiheptide U (NOS-U), a new NOS-type compound with an extra hydroxyl group at the indole nitrogen, we report that NocU is responsible for the N-hydroxylation of the indolic moiety during the maturation of NOCs. This finding reveals the cause of structural differences at the indole nitrogen of NOCs, which will not only accelerate the biosynthetic studies of NOCs, but also promote new analog development by utilizing the compatibility of the biosynthetic machinery of thiopeptide antibiotics.