BIOSYNTHESIS OF TAXOL

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Taxol, a highly functionalized diterpenoid, is an important antitumor drug isolated from the Western Yew, Taxus brevifolia, and other Taxus species. The supply of this drug from natural sources is very limited. Any attempt to improve the biological production of taxol requires an understanding of the biosynthesis of this natural product and of the regulation of the pathway. A biogenetic scheme has been proposed based on the occurrence of defined taxane metabolites and on analogy to biosynthetic transformations of simpler monoterpenoids; however, there is at present no experimentally supported information on the biosynthesis of taxol. The objective of this project is to determine the number, types and sequence of enzymatic steps in the transformation of the C20 isoprenoid branch-point intermediate, geranylgeranyl pyrophosphate, to taxol, and to determine the rate limiting step(s) of the pathway. Specific aims include: (1) defining the cyclization step of geranylgeranyl pyrophosphate and the identity of the olefin precursor of taxol; (2) establishing the sequence of hydroxylations of the parent olefin on route to taxol, and deciphering the sequence of acetylations of the hydroxyl groups and the possible biosynthetic function of the acetylation process; (3) elucidating the enzymatic route of oxetane ring formation; (4) determining the order and mechanism of late stage acylations at C-2 and C-13 of the taxol nucleus; and (5) confirming the pathway by in vivo studies and delineating the rate limiting step(s) of the pathway. This goal of describing the complex pathway to taxol will be accomplished through the use of cell-free enzyme systems from T. brevifolia and the use of radiolabeled precursors. Radiolabeled products from the early enzymatic reactions will be employed to direct the isolation and identification of the corresponding intermediates from Taxus extracts, from which labeled substrates for succeeding enzymatic transformations will be prepared. In vivo studies employing 14CO2 labeling in intact saplings will then be used to confirm the pathway and, in combination with isotopic dilution analysis, to determine the rate limiting step(s). The rate limiting catalyst, and other enzymes of the pathway relevant to semi-synthetic efforts to produce taxol from more readily available taxane metabolites, will be the focus of more detailed study as the foundation for genetic and molecular approaches to the improved in vivo or in vitro production of the target compound.