A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis

Area of Science:

• Biochemistry
• Enzymology
• Natural Product Biosynthesis

Background:

• Tetracyclic taxoids are complex natural products with significant pharmaceutical applications.
• Paclitaxel biosynthesis involves intricate enzymatic steps, including the formation of unique structural motifs.
• Cytochrome P450 enzymes play diverse roles in the metabolism of natural products.

Purpose of the Study:

• To functionally characterize oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme.
• To elucidate the enzymatic mechanism of oxetane ring formation in tetracyclic taxoids.
• To understand the role of TmCYP1 in the biosynthesis pathway of paclitaxel.

Main Methods:

• Transient expression of TmCYP1 in Nicotiana benthamiana.
• Substrate-feeding experiments with specific taxoid derivatives.
• Isotope labeling studies (2H and 18O) and density functional theory calculations.

Main Results:

• TmCYP1 efficiently catalyzed the formation of an oxetane ring from a hexaacetoxytaxadiene substrate.
• A 4β,20-epoxide derivative was identified as a major or sole product when using a 5-deacetylated substrate.
• The C5-O-acetyl group was found to be essential for oxetane ring formation but not for epoxide formation.

Conclusions:

• TmCYP1 is a key enzyme in the formation of the oxetane ring during paclitaxel biosynthesis.
• The reaction mechanism involves intramolecular oxidation-acetoxyl rearrangement, potentially via a concerted process.
• The findings provide critical insights into the enzymatic strategies for constructing the oxetane moiety in taxoids.