PURIFICATION AND CHARACTERIZATION OF TAXA-4(5),11(12)-DIENE SYNTHASE FROM PACIFIC YEW (TAXUS-BREVIFOLIA) THAT CATALYZES THE FIRST COMMITTEDSTEP OF TAXOL BIOSYNTHESIS

The first step in the biosynthesis of taxol in Pacific yew (Taxus brev ifolia) is the cyclization of the universal diterpene precursor gerany lgeranyl pyrophosphate to taxa-4(5),11(12)-diene, This parent olefin o f the taxane diterpenoids is then elaborated to taxol and related comp ounds by a complex series of reactions involving oxidations and side-c hain acylations. Cyclization activity is located principally in yew st em bark and adhering cambium, The operationally soluble cyclization en zyme was partially purified (similar to 600-fold) by combination of an ion exchange, hydrophobic interaction, and dye-ligand chromatography. Nondenaturing, followed by denaturing, polyacrylamide gel electrophore sis, in combination with gel permeation chromatography, allowed the id entification of taxadiene synthase as a monomeric protein of molecular weight 79,000. In general properties (divalent metal ion requirement, kinetic constants, molecular weight), the taxadiene synthase of Pacif ic yew is similar to the diterpene cyclase abietadiene synthase involv ed in resin acid biosynthesis in other gymnosperms. However, in pH opt imum and response to inhibitors, these two diterpene cyclases are dist inctly different. The activity (and enzyme protein) levels of Pacific yew taxadiene synthase are much lower than those for abietadiene synth ase of lodgepole pine stem (constitutive) or of grand fir stem (wound- inducible) and the enzyme is not inducible to higher levels by stem wo unding or elicitor treatment. (C) 1995 Academic Press, Inc.