A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis
Changkang Li, Xinxin Yin, Shuai Wang, Songyang Sui, Jimei Liu, Xincheng Sun, Jinming Di, Ridao Chen, Dawei Chen, Yaotian Han, Kebo Xie, Kebo Xie, Jungui Dai
Abstract
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.