Cytochrome P450-Catalyzed Tetrahydrofuran Formation via Dual Pathways in Avermectin Biosynthesis

Avermectins (AVMs) are a class of 16-membered ring macrolides produced by Streptomyces avermitilis. Renowned for their potent insecticidal and acaricidal properties, AVMs are widely used as environmentally friendly biopesticides. Although the biosynthetic gene aveE encoding a cytochrome P450 monooxygenase was identified 30 years ago, its exact catalytic function and mechanism have remained elusive due to a lack of biochemical characterization. Here, we overcome the long-standing challenge in soluble and functional protein expression of AveE in Escherichia coli and reconstitute the in vitro activity of this P450 enzyme using surrogate redox partner proteins. Time-course studies reveal monohydroxylation at the C8a position of the substrates as the initial step, subsequently leading to tetrahydrofuran (THF) ring formation. Isotopic labeling experiments provide significant insight into the catalytic mechanism of AveE, revealing the cooperation of the C8a,C6-diol pathway and the C6-nucleophilic attack pathway. This study not only presents an effective strategy for heterologous expression of difficult P450 enzymes in E. coli but also elucidates the exact process of THF ring formation during AVM biosynthesis.