Publications

• Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis.

  Koksal M., Jin Y., Coates R.M., Croteau R., Christianson D.W.

  With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin poly ... >> More

  With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 Å resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 Å resolution). The TXS structure reveals a modular assembly of three α-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third 'insertion' domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis. << Less

  Nature 469:116-120(2011) [PubMed] [EuropePMC]

• Purification and characterization of taxa-4(5),11(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first committed step of taxol biosynthesis.

  Hezari M., Lewis N.G., Croteau R.

  The first step in the biosynthesis of taxol in Pacific yew (Taxus brevifolia) is the cyclization of the universal diterpene precursor geranylgeranyl pyrophosphate to taxa-4(5),11(12)-diene. This parent olefin of the taxane diterpenoids is then elaborated to taxol and related compounds by a complex ... >> More

  The first step in the biosynthesis of taxol in Pacific yew (Taxus brevifolia) is the cyclization of the universal diterpene precursor geranylgeranyl pyrophosphate to taxa-4(5),11(12)-diene. This parent olefin of the taxane diterpenoids is then elaborated to taxol and related compounds by a complex series of reactions involving oxidations and side-chain acylations. Cyclization activity is located principally in yew stem bark and adhering cambium. The operationally soluble cyclization enzyme was partially purified (approximately 600-fold) by combination of anion exchange, hydrophobic interaction, and dye-ligand chromatography. Nondenaturing, followed by denaturing, polyacrylamide gel electrophoresis, in combination with gel permeation chromatography, allowed the identification 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 Pacific yew is similar to the diterpene cyclase abietadiene synthase involved in resin acid biosynthesis in other gymnosperms. However, in pH optimum and response to inhibitors, these two diterpene cyclases are distinctly different. The activity (and enzyme protein) levels of Pacific yew taxadiene synthase are much lower than those for abietadiene synthase of lodgepole pine stem (constitutive) or of grand fir stem (wound-inducible) and the enzyme is not inducible to higher levels by stem wounding or elicitor treatment. << Less

  Arch Biochem Biophys 322:437-444(1995) [PubMed] [EuropePMC]

• Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures.

  Hezari M., Ketchum R.E., Gibson D.M., Croteau R.

  The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene represents the first committed, and a slow, step in the complex biosynthetic pathway leading to the anticancer drug Taxol. The cyclization enzyme, taxadiene synthase, has been previously purified from Pacific yew (Taxus brevif ... >> More

  The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene represents the first committed, and a slow, step in the complex biosynthetic pathway leading to the anticancer drug Taxol. The cyclization enzyme, taxadiene synthase, has been previously purified from Pacific yew (Taxus brevifolia) stem and characterized, and the corresponding cDNA has been isolated. To better assess the role of taxadiene synthase in the control of pathway flux in Canadian yew (T. canadensis) cells, a reliable system for production of Taxol in suspension culture, the enzyme from this source was isolated and shown to be chromatographically, electrophoretically, and kinetically identical to that of T. brevifolia stem. Results from the analysis of enzyme activity levels during the time course of Taxol accumulation in developing cell cultures of T. canadensis indicate that rate-limiting transformations lay farther down the pathway than the cyclization step in this system. << Less

  Arch Biochem Biophys 337:185-190(1997) [PubMed] [EuropePMC]

• Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxadiene precursor of taxol catalyzed by recombinant taxadiene synthase.

  Williams D.C., Carroll B.J., Jin Q., Rithner C.D., Lenger S.R., Floss H.G., Coates R.M., Williams R.M., Croteau R.

  <h4>Background</h4>The committed step in the biosynthesis of the anticancer drug taxol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this complex olefin cation cyclization cascade involving the formation o ... >> More

  <h4>Background</h4>The committed step in the biosynthesis of the anticancer drug taxol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this complex olefin cation cyclization cascade involving the formation of three rings and three stereogenic centers.<h4>Results</h4>Recombinant taxadiene synthase was incubated with specifically deuterated substrates, and the mechanism of cyclization was probed using MS and NMR analyses of the products to define the crucial hydrogen migration and terminating deprotonation steps. The electrophilic cyclization involves the ionization of the diphosphate with closure of the A-ring, followed by a unique intramolecular transfer of the C11 proton to the re-face of C7 to promote closure of the B/C-ring juncture, and cascade termination by proton elimination from the beta-face of C5.<h4>Conclusions</h4>These findings provide insight into the molecular architecture of the first dedicated step of taxol biosynthesis that creates the taxane carbon skeleton, and they have broad implications for the general mechanistic capability of the large family of terpenoid cyclization enzymes. << Less

  Chem Biol 7:969-977(2000) [PubMed] [EuropePMC]

• Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene is the committed step of taxol biosynthesis in Pacific yew.

  Koepp A.E., Hezari M., Zajicek J., Vogel B.S., LaFever R.E., Lewis N.G., Croteau R.

  The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems ca ... >> More

  The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems catalyzed the conversion of [1-3H]geranylgeranyl diphosphate to a cyclic diterpene olefin that, when incubated with stem sections, was converted in good radiochemical yield to several highly functionalized taxanes, including 10-deacetyl baccatin III and taxol itself. Addition of the labeled olefin to a yew bark extract, followed by radiochemically guided fractionation, provided sufficient product to establish the structure as taxa-4(5),11(12)-diene by two-dimensional NMR spectroscopic methods. Therefore, the first dedicated step in taxol biosynthesis is the conversion of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11(12)-diene, rather than to the 4(20),11(12)-diene isomer previously suggested on the basis of the abundance of taxoids with double bonds in these positions. The very common occurrence of taxane derivatives bearing the 4(20)-ene-5-oxy functional grouping, and the lack of oxygenated derivatives bearing a 4(5)-double bond, suggest that hydroxylation at C-5 of taxadiene with allylic rearrangement of the double bond is an early step in the conversion of this olefin intermediate to taxol. << Less

  J Biol Chem 270:8686-8690(1995) [PubMed] [EuropePMC]

• Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction.

  Williams D.C., Wildung M.R., Jin A.Q., Dalal D., Oliver J.S., Coates R.M., Croteau R.

  The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence ... >> More

  The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids. Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired. Since the transit peptide-mature enzyme cleavage site could not be determined directly, a series of N-terminally truncated enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional protein; however, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme. In addition to the major product, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%). Isotopically sensitive branching experiments utilizing (4R)-[4-(2)H(1)]geranylgeranyl diphosphate confirmed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the enzyme to utilize 2, 7-cyclogeranylgeranyl diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure). Two potential mechanism-based inhibitors were tested with recombinant taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition. << Less

  Arch Biochem Biophys 379:137-146(2000) [PubMed] [EuropePMC]

• Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in Pacific yew.

  Lin X., Hezari M., Koepp A.E., Floss H.G., Croteau R.

  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)-die ... >> More

  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. << Less

  Biochemistry 35:2968-2977(1996) [PubMed] [EuropePMC]