| The highly functionalized diterpenoid Taxol, derived from yew ( Taxus) species, is a potent antineoplastic drug used successfully in the treatment of a wide variety of cancers. The limited natural source of this drug has prompted the development of alternative production methods, such as semisynthesis from more available taxoid precursors isolated from needles of various Taxus species, which serve as the current source of Taxol. Thus, for the foreseeable future, the supply of Taxol must continue to rely upon biological methods of production and improvements to this biosynthetic process in yew or derived cell cultures requires a full understanding of the Taxol/taxoid biosynthetic pathways.; The Taxol biosynthetic pathway consists of approximately 20 enzymatic steps from the primary intermediate geranylgeranyl diphosphate, which is cyclized to taxa-4(5),11(12)-diene. This parental olefin then undergoes a series of at least nine cytochrome P450-mediated oxygenations, five acylations, and several other modifications en route to Taxol. To probe for mid-pathway cytochrome P450 hydroxylases, a 'surrogate' substrate, taxusin (taxadien-5alpha,9alpha,10beta,13alpha-tetraol tetraacetate), was employed to functionally screen a family of cytochrome P450 oxygenases originating from Taxus cell cDNA libraries. This screen led to the identification of a taxoid 2alpha-hydroxylase and a taxoid 7beta-hydroxylase, both of which are capable of converting taxusin to their respective derivatives, as well as the reciprocal conversion of their respective pentaol tetraacetate products to a common hexaol tetraacetate.; Additionally, a comparable functional screen using polyhydroxylated substrates was performed with similarly isolated Taxus acyltransferase clones, which led to the identification of another taxoid 5alpha- O-acetyltransferase. However, this enzyme demonstrated several differences from the previously-isolated 5alpha-O-acetyltransferase; both are capable of converting taxadien-5alpha-ol to taxadien-5alpha-yl acetate but display different regiospecificity with polyhydroxylated taxoid substrates. Examination of kinetic profiles suggests that the previously-isolated 5alpha-O-acetyltransferase prefers to acetylate taxadien-5alpha-ol, whereas this new acetyltransferase has a broader specificity and may be responsible for the abundance of acetylated taxoids.; The isolation of these enzymes and their corresponding genes is important in gaining an understanding of Taxol biosynthesis. Also, manipulation of these genes could allow increased production of Taxol, as well as the generation of other useful taxoid derivatives for subsequent studies. |
