Nosiheptide and nocathiacin are thiopeptides within the same series with high similarity from chemical structure to organization of the biosynthetic gene cluster. Previously, NosB, a cytochrome P450-like enzyme, was demonstrated to be responsible for the hydroxylation of Glu-6 during nosiheptide maturation. Based on 64% amino acid sequence identity, NocB from Nocardia sp. ATCC 202099 is expected to exhibit a similar catalytic function to NosB. After replacing nosB by nocB in nosiheptide-producing Streptomyces actuosus ATCC 25421, NocB was proved to be a cytochrome P450-like monooxygenase responsible for the hydroxylation of Glu-6 at the γ-position in the biosyntheses of both nosiheptide and nocathiacin. Enzyme kinetics and structural difference between nosiheptide and nocathiacin further revealed that NocB is less active than NosB towards unglycosylated intermediate 3 containing a bicyclic core structure.