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.
Identifying beneficial distal mutations remains a key challenge in enzyme engineering, as such residues can regulate activity through long-range dynamical coupling and allosteric communication. Although protein language models effectively capture sequence and evolutionary information, they lack explicit representation of conformational dynamics in the presence of the substrate, limiting their ability to detect distal regulatory sites. Here, we present an integrated framework that combines molecular dynamics (MD)-derived descriptors with the zero-shot prediction model GEMS to identify beneficial distal mutations. Compared to the pure zero-shot model, MD-derived descriptors efficiently capture key distal residues involved in dynamical coupling and allosteric communication with the active site. Consequently, these constraints enable the zero-shot model to predict distal mutations more precisely. By integrating sequence, evolutionary, and dynamic information, our approach expands the diversity of candidate sites while maintaining a manageable screening scale, offering an efficient and generalizable strategy for enzyme engineering.
Yiqiu Wang, Ding Luo, Shuming Cheng et al.· Journal of Chemical Theory a...· 0 citations