title: Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in Pacific yew.
abstract: The first committed step in the formation of taxol has been shown to involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The formation of this endocyclic diterpene olefin isomer as the precursor of taxol was unexpected, since the exocyclic isomer, taxa-4(20),11(12)-diene, had been predicted as the initial product of the taxol pathway on the basis of metabolite co-occurrence. [1-2H2,20-2H3] and [20-2H3]geranylgeranyl diphosphates were employed as substrates with the partially purified taxadiene synthase from Pacific yew (Taxus brevifolia) stems to examine the possibility of a preliminary 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 parent ion and the P-15 and C-ring fragment ions, compared to those of unlabeled standard, showed the olefin product to possess a deuterium enrichment essentially identical to that of the acyclic precursor, thus ruling out the putative isomerization step. With [4-2H2]geranylgeranyl diphosphate as substrate, similar product analysis established the enzymatically derived taxa-4(5),11(12)-diene to contain only one deuterium 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 these exogenous olefins could serve as intermediates of the cyclization reaction. However, GLC-MS analysis of the taxadiene product derived by enzymatic cyclization of [1-3H]geranylgeranyl diphosphate in 2H2O indicated little incorporation of deuterium from the medium and suggested a rapid internal proton transfer in a tightly bound olefinic intermediate. Analysis of the enzymatic product generated from [10-2H1]geranylgeranyl diphosphate confirmed the intramolecular hydrogen transfer from C11 of a verticillyl intermediate to the C-ring of taxa-4(5),11(12)-diene. From these results, a stereochemical mechanism is proposed for the taxadiene synthase reaction involving the initial cyclization of geranylgeranyl diphosphate to a transient verticillyl cation intermediate, with transfer of the C11 alpha-proton to C7 to initiate transannular B/C-ring closure to the taxenyl cation, followed by deprotonation at C5 to yield the taxa-4(5),11(12)-diene product directly.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, USA.
authors.forename: X
authors.initials: X
authors.lastname: Lin
authors.forename: M
authors.initials: M
authors.lastname: Hezari
authors.forename: A E
authors.initials: AE
authors.lastname: Koepp
authors.forename: H G
authors.initials: HG
authors.lastname: Floss
authors.forename: R
authors.initials: R
authors.lastname: Croteau
mesh_terms: D007535:Isomerases / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D017239:Paclitaxel / Q000096:biosynthesis*; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D017368:Protein Prenylation; D011865:Radioisotope Dilution Technique; D014197:Trees / Q000201:enzymology; D014316:Tritium
chemical_list: D011106:Polyisoprenyl Phosphates; D014316:Tritium; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase; C002963:geranylgeranyl pyrophosphate; D017239:Paclitaxel