MECHANISM OF TAXADIENE SYNTHASE, A DITERPENE CYCLASE THAT CATALYZES THE FIRST STEP OF TAXOL BIOSYNTHESIS IN PACIFIC YEW

The first committed step in the formation of taxol has been shown to i nvolve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(1 2)-diene. The formation of this endocyclic diterpene olefin isomer as the precursor of taxol was unexpected, since the exocyclic isomer. tax a-4(20),11(12)-diene, had been predicted as the initial product of the taxol pathway on the basis of metabolite co-occurrence. [1-H-2(2),20- H-2(3)] and [20-H-2(3)]geranylgeranyl diphosphates were employed as su bstrates with the partially purified taxadiene synthase from Pacific y ew (Taxus brevifolia) stems to examine the possibility of a preliminar y cyclization to taxa-4(20),11(12)-diene followed by isomerization to the more stable endocyclic double bond isomer. GLC-MS analysis of the derived taxa-4(5),11(12)-diene, via selected ion monitoring of the par ent ion and the P-15 and C-ring fragment ions, compared to those of un labeled standard? showed the olefin product to possess a deuterium enr ichment essentially identical to that of the acyclic precursor, thus r uling out the putative isomerization step, With [4-H-2(2)]geranylgeran yl diphosphate as substrate, similar product analysis established the enzymatically derived taxa-4(5). 11(12)-diene to contain only one deut erium atom, consistent with direct formation from a taxenyl cation by deprotonation at C5. (+/-)-Casbene, (+/-)-verticillene, and (+/-)-taxa -4(20), 11(12)-diene were tested as possible olefinic intermediates in taxa-4(5), 11(12)-diene formation by a series of inhibition, trapping , and direct conversion experiments; no evidence was obtained that the se exogenous olefins could serve as intermediates of the cyclization r eaction. However, GLC-MS analysis of the taxadiene product derived by enzymatic cyclization of [1-H-3]geranylgeranyl diphosphate in (H2O)-H- 2 indicated little incorporation of deuterium from the medium and sugg ested a rapid internal proton transfer in a tightly bound olefinic int ermediate, Analysis of the enzymatic product generated from [10-H-2(1) ]geranylgeranyl diphosphate confirmed the intramolecular hydrogen tran sfer from C11 of a verticillyl intermediate to the C-ring of taxa-4(5) ,11(12)-diene. From these results, a stereochemical mechanism is propo sed for the taxadiene synthase reaction involving the initial cyclizat ion of geranylgeranyl diphosphate to a transient verticillyl cation in termediate, with transfer of the CI 1 or-proton to C7 to initiate tran sannular B/C-ring closure to the taxenyl cation, followed by deprotona tion at C5 to yield the taxa-4(5),11(12)-diene product directly.