Mechanism of Taxadiene Synthase, a Diterpene Cyclase That Catalyzes the First Step of Taxol Biosynthesis in Pacific Yew

• Published in: Biochemistry (Easton) Vol. 35; no. 9; pp. 2968 - 2977
• Main Authors: Lin, Xiaoyan, Hezari, Mehri, Koepp, Alfred E, Floss, Heinz G, Croteau, Rodney
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05.03.1996
• Subjects: Isomerases - metabolism; Isotope Labeling - methods; Magnetic Resonance Spectroscopy; Molecular Structure; Paclitaxel - biosynthesis; Polyisoprenyl Phosphates - chemical synthesis; Polyisoprenyl Phosphates - chemistry; Polyisoprenyl Phosphates - metabolism; Protein Prenylation; Radioisotope Dilution Technique; Trees - enzymology; Tritium
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9526239

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 α-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.