Cytochrome P450 enzymes in the biosynthesis of bioactive diterpenoids from traditional chinese medicinal plants.

Cytochrome P450 enzymes play key catalytic roles in the biosynthesis of diterpenoid natural products from traditional Chinese medicinal plants. Operating through a conserved heme-thiolate catalytic cycle and Compound I-driven oxidation, these enzymes mediate most post-cyclization oxidative tailoring of diterpene scaffolds, including hydroxylation, epoxidation, carbon-skeleton rearrangement, and lactone formation. This review surveys the biosynthetic pathways of seven representative diterpenoids, namely tanshinones, paclitaxel, triptolide, andrographolide, ginkgolides, forskolin, and oridonin, in which sequential site-selective hydroxylation and multifunctional catalysis are recurring features, whereas carbon-skeleton remodeling proceeds through pathway-specific modes, including Wagner-Meerwein methyl migration in triptolide, NIH-shift alkyl migration and dual C-C bond cleavage in ginkgolides, and oxidative oxetane-ring formation in paclitaxel, with lactone ring construction observed in the andrographolide, triptolide, and ginkgolide pathways. Atomic-level studies of representative enzymes CYP76AH1, CYP725A4, TOT1, and TmCYP1 further clarify how substrate recognition, regioselectivity, and catalytic trajectories are governed within the active site, providing a basis for rational enzyme engineering. Finally, the prospects of artificial intelligence-assisted protein design for engineering P450 catalytic elements are discussed, encompassing structure prediction, protein language model-based mutational prescreening, and de novo catalytic-pocket design, along with their potential to facilitate heterologous biosynthesis and the sustainable production of bioactive diterpenoids from medicinal plants.