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

A central feature in the biosynthesis of Taxol is oxygenation at multiple p ositions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based different ial display-cloning approach, using Taxus (yew) cells induced for Taxol pro duction, yielded a family of related cytochrome P450 genes, one of which wa s assigned as a taxane 10 beta -hydroxylase by functional expression in yea st. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and we re screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alph a -ol and its acetate ester as test substrates. This approach allowed ident ification of one of the cytochrome P450 clones (which bore 63% deduced sequ ence identity to the aforementioned taxane 10 beta -hydroxylase) as a taxan e 13 alpha -hydroxylase by chromatographic and spectrometric characterizati on of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 gene s validates this strategy for elucidating the oxygenation steps of taxane d iterpenoid (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.