CYTOCHROME-P450-CATALYZED HYDROXYLATION OF TAXA-4(5),11(12)-DIENE TO TAXA-4(20),11(12)-DIEN-5-ALPHA-OL - THE FIRST OXYGENATION STEP IN TAXOL BIOSYNTHESIS

Background: The structural complexity of taxol dictates continued reli ance on biological production methods, which may be improved by a deta iled understanding of taxol biosynthesis, especially the rate-limiting steps, The biosynthesis of taxol involves the cyclization of the comm on isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5), 11 (12)-diene followed by extensive, largely oxidative, modification of t his diterpene olefin. We set out to define the first oxygenation step in taxol biosynthesis. Results: Microsomal enzymes from Taxus stem and cultured cells were used to define the first hydroxylation of taxadie ne. We confirmed the structure of the reaction product (taxa-4(20), 11 (12)-dien-5 alpha-ol) by synthesizing this compound. The responsible b iological catalyst was characterized as a cytochrome P450 theme thiola te protein). In vivo studies confirmed that taxadienol is a biosynthet ic intermediate and indicated that the hydroxylation step that produce s this product is stow relative to subsequent metabolic transformation s. Conclusions: The structure of the first oxygenated intermediate on the taxol pathway establishes that the hydroxylation reaction proceeds with an unusual double bond migration that limits the mechanistic pos sibilities for subsequent elaboration of the oxetane moiety of taxol, The reaction is catalyzed by a cytochrome P450, suggesting that the se ven remaining oxygenation steps in taxol biosynthesis may involve simi lar catalysts, Because the first oxygenation step is slow relative to subsequent metabolic transformations, it may be possible to speed taxo l biosynthesis by isolating and manipulating the gene for the taxadien e-5-hydroxylase that catalyzes this reaction.