A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis

Abstract Oxetane synthase ( Tm CYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2 α ,5 α ,7 β ,9 α ,10 β ,13 α ‐hexaacetoxytaxa‐4(20),11(12)‐diene ( 1 ) as a substrate led to the production of a major oxetane derivative, 1 β ‐dehydroxybaccatin IV ( 1 a ), and a minor 4 β ,20‐epoxide derivative, baccatin I ( 1 b ). However, feeding the substrate decinnamoyltaxinine J ( 2 ), a 5‐deacetylated derivative of 1 , yielded only 5 α ‐deacetylbaccatin I (2 b ), a 4 β ,20‐epoxide. A possible reaction mechanism was proposed on the basis of substrate‐feeding, 2 H and 18 O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation‐acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4 β ,20‐epoxide might be a shunt product. In this process, the C5‐ O ‐acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)‐epoxy ring formation by Tm CYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.