Cytochrome p450 taxadiene 5alpha-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis.

Abstract

The first oxygenation step in the biosynthesis of the anticancer drug taxol in Taxus species is the cytochrome p450-mediated hydroxylation (with double bond migration) of the diterpene olefin precursor taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol. A homology-based cloning strategy, employing an induced Taxus cell library, yielded a cDNA encoding taxadiene 5alpha-hydroxylase, which was functionally expressed in yeast and insect cells. The recombinant enzyme was characterized and shown to efficiently utilize both taxa-4(5),11(12)-diene and taxa-4(20),11(12)-diene (as an adventitious substrate) to synthesize taxa-4(20),11(12)-dien-5alpha-ol. This hydroxylase resembles, in sequence and properties, other cytochrome p450 oxygenases of taxol biosynthesis. The utilization of both taxadiene isomers in the formation of taxa-4(20),11(12)-dien-5alpha-ol is novel, suggesting a reaction mechanism involving promiscuous radical abstraction with selective oxygen insertion rather than epoxidation of the C4,C5-alkene of the natural substrate and allylic rearrangement of the resulting taxa-11(12)-en-4,5epoxide.