Stefan Jennewein

Taxol biosynthesis: Taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10β-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5α-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10β-hydroxylase) as a taxane 13α-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.

Taxoid metabolism: Taxoid 14β-hydroxylase is a cytochrome P450-dependent monooxygenase☆☆☆

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.