Promiscuity of Cytochrome P450 Enzymes: Discovery Strategies, Mechanistic Insights, and Applications in Terpenoid Biosynthesis

Enzyme promiscuity drives metabolic diversity and evolutionary innovation. Among these, the cytochrome P450 (CYP) superfamily plays a central role in generating the immense structural complexity of terpenoid natural products. This review summarizes recent advances in understanding CYP substrate promiscuity, with a focus on discovery strategies, mechanistic foundations, and applications in terpenoid biosynthesis. We first highlight high-throughput mining and expression systems for CYP identification, alongside protein engineering to expand catalytic scope. Mechanistically, we integrate insights into active-site dynamics, electron-transfer partner compatibility, and evolutionary processes such as gene duplication and neofunctionalization to explain broad substrate acceptance and multistep oxidation reactions. Finally, representative promiscuous P450 enzymes from plants, bacteria, and fungi are summarized. These findings highlight both the opportunities and current limitations of exploiting CYP promiscuity for the diversification of agriculturally and nutritionally relevant terpenoids.