A Cytochrome P450 Enzyme-Catalyzed Acetyl Migration in Cyanogramide Biosynthesis

Cyanogramide (1) is a unique spirooxindole alkaloid derived from a marine actinomycete and is characterized by its distinct spirocyclic pyrrolo[1,2-c]imidazolidin-4-one scaffold. Although we have successfully elucidated the biosynthetic pathway of 1, the mechanism underlying the formation of the characteristic imidazolidin-4-one remains unclear. In this study, we demonstrate that the cytochrome P450 monooxygenase CyaI catalyzes an oxidation reaction through a zwitterionic intermediate and facilitates a subsequent unusual C→N acetyl migration, which triggers a spontaneous intramolecular cyclization to forge the imidazolidin-4-one ring during 1 biosynthesis. In addition, CyaI is identified as a bifunctional enzyme that also catalyzes N-demethylation. High-resolution crystallography and mutagenesis studies determine Thr245 as a crucial catalytic residue that modulates the balance between imidazolidine-4-one synthesis and demethylation. This work not only expands the catalytic repertoire of P450 enzymes but also opens the way for the development of multifunctional biocatalysts in the synthesis of complex natural products.