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Abietadiene

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Rodney Croteau – 1st expert on this subject based on the ideXlab platform

  • bifunctional Abietadiene synthase mutual structural dependence of the active sites for protonation initiated and ionization initiated cyclizations
    Biochemistry, 2003
    Co-Authors: Reuben J Peters, Ora A Carter, Yan Zhang, Brian W Matthews, Rodney Croteau

    Abstract:

    Abietadiene synthase from grand fir catalyzes two sequential, mechanistically distinct cyclizations, of geranylgeranyl diphosphate and of copalyl diphosphate, in the formation of a mixture of Abietadiene isomers as the committed step of diterpenoid resin acid biosynthesis. Each reaction is independently conducted at a separate active site residing in what were considered to be structurally distinct domains typical of terpene cyclases. Despite the presence of an unusual 250-residue N-terminal insertional element, a tandem pair of charged residues distal to the insertion was shown to form a functional part of the C-terminal active site. Because Abietadiene synthase resembles the ancestral plant terpene cyclase, this observation suggests an early evolutionary origin of catalytically important positively charged residues at the N-terminus of enzymes of this general class. A series of N- and C-terminal truncations of this enzyme were constructed and characterized, both alone and as mixtures of adjacent polypep…

  • Diterpenoid Resin Acid Biosynthesis in Conifers: Characterization of Two Cytochrome P450-Dependent Monooxygenases and an Aldehyde Dehydrogenase Involved in Abietic Acid Biosynthesis
    Archives of Biochemistry and Biophysics, 2002
    Co-Authors: C Funk, Rodney Croteau

    Abstract:

    Abstract Abietic acid is a major component of the rosin fraction of oleoresin synthesized by grand fir (Abies grandis), lodgepole pine (Pinus contorta), and many other conifer species as a defensive secretion against insect and pathogen attack. The diterpenoid resin acid is derived from geranylgeranyl pyrophosphate via Abietadiene, with subsequent oxidation of the C18-methyl of this olefin to abietadienol, abietadienal, and abietic acid. The pathway was confirmed by administering [1,2-14C)acetic acid to grand fir stems which incorporated the radiolabel into Abietadiene, the corresponding alcohol and aldehyde, as well as abietic acid. Three different enzymatic activities, catalyzing the sequential oxidation of the olefin to abietic acid, were demonstrated in cell-free stem extracts of both grand fir and lodgepole pine. The first two oxidation steps were catalyzed by the microsomal fraction and required both oxygen and a reduced pyridine nucleotide (NADPH preferred). Both activities were strongly inhibited by CO (blue light reversible) and were differentially sensitive to several substituted N-heterocyclic inhibitors, suggesting that these two enzymes are distinct, microsomal cytochrome P450-dependent monooxygenases. A third enzymatic activity, catalyzing the oxidation of abietadienal to abietic acid, was located in the soluble protein fraction. This oxidation reaction employed NAD+ as cofactor, but did not require oxygen and was not inhibited by CO, indicating that this last step of abietic acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase.

  • mechanism of Abietadiene synthase catalysis stereochemistry and stabilization of the cryptic pimarenyl carbocation intermediates
    Journal of the American Chemical Society, 2002
    Co-Authors: Matthew M Ravn, Robert M Coates, Reuben J Peters, Rodney Croteau

    Abstract:

    Abietadiene synthase (AS) catalyzes the complex cyclization−rearrangement of (E,E,E)-geranylgeranyl diphosphate (8, GGPP) to a mixture of Abietadiene (1a), double bond isomers 2a−4a and pimaradienes 5a−7a as a key step in the biosynthesis of the abietane resin acid constituents (1b−4b) of conifer oleoresin. The reaction proceeds at two active sites by way of the intermediate, copalyl diphosphate (9). In the second site, a putative tricyclic pimaradiene or pimarenyl(+) carbocation intermediate of undefined C13 stereochemistry and annular double bond position is formed. Three 8-oxy-17-nor analogues of 9 (17 and 19a,b) and three isomeric 15,16-bisnorpimarenyl-N-methylamines (26a−c) were synthesized and evaluated as alternative substrates and/or inhibitors for recombinant AS from grand fir. The stereospecific cyclization of 8α-hydroxy-17-nor CPP (19a) to 17-normanoyl oxide (20a) and the higher inhibitory potency of the norpimarenylamine 26a (Ki = 0.1 nM) both suggest pimarenyl intermediates having the 13β met…

Jorg Bohlmann – 2nd expert on this subject based on the ideXlab platform

  • Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional
    , 2020
    Co-Authors: Dawn E. Hall, Philipp Zerbe, Sharon Jancsik, Alfonso Lara Quesada, Harpreet K. Dullat, Lufiani L. Madilao, Macaire Yuen, Jorg Bohlmann

    Abstract:

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/Abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs. Conifer trees, including lodgepole pine (Pinus contorta) and jack pine (Pinus banksiana), produce complex mixtures of mono-, sesqui-, and diterpenoid specialized (i.e. secondary) metabolites, most prominently in the form of oleoresin, that can act as a physical and chemical defense against insect and pathogen attack (Phillips and Croteau, 1999; Keeling and Bohlmann, 2006a, 2006b; Zulak and Bohlmann, 2010; Boone et al., 2011). These oleoresin terpenoids also serve as a large-volume, renewable resource for industrial bioproducts, including solvents, flavors, fragrances,

  • Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its
    , 2020
    Co-Authors: Philipp Zerbe, Macaire Yuen, Jorg Bohlmann, Angela Chiang, Björn Hamberger, Jason Draper, Robert A. Britton, Canadaandthe § Departmentofchemistry

    Abstract:

    The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/Abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/Abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunc

  • immunofluorescence localization of levopimaradiene Abietadiene synthase in methyl jasmonate treated stems of sitka spruce picea sitchensis shows activation of diterpenoid biosynthesis in cortical and developing traumatic resin ducts
    Phytochemistry, 2010
    Co-Authors: Katherine G Zulak, Harpreet K. Dullat, Christopher I Keeling, Dustin Lippert, Jorg Bohlmann

    Abstract:

    Abstract Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species ( Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by insects, fungi or treatment with methyl jasmonate (MeJA). Using immunofluorescence localization we tested if previously formed CRDs respond to MeJA treatment with increased capacity for diterpenoid biosynthesis. We also tested the dynamics of diterpene synthase localization in the cambial zone. Immunofluorescence localization was performed using an antibody against a diterpene synthase, levopimaradiene/Abietadiene synthase (LAS), in stem cross-sections of untreated and 0.1% MeJA-treated 4-year old Sitka spruce ( P. sitchensis ) trees. No fluorescence signal was observed in untreated stem cross-sections; however, signal was present 2 days after treatment with MeJA exclusively in the epithelial cells of CRDs. Fluorescence steadily increased in the CRD epithelial cells 4 and 8 days after treatment. At 8 days, additional fluorescence was observed in developing epithelial cells of traumatic resin ducts TRDs in the cambial zone. These results confirm that resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in CRD epithelial cells early upon treatment with MeJA, and immature developing TRD epithelial cells produce diterpene synthase enzyme. Overall, the results of this work improve our understanding of spatial and temporal patterns of induced diterpene resin acid biosynthesis in conifers.

Reuben J Peters – 3rd expert on this subject based on the ideXlab platform

  • a single residue change leads to a hydroxylated product from the class ii diterpene cyclization catalyzed by Abietadiene synthase
    Organic Letters, 2012
    Co-Authors: Jared Criswell, Kevin C Potter, Freya Shephard, Michael H Beale, Reuben J Peters

    Abstract:

    Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of Abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity.

  • insights into diterpene cyclization from structure of bifunctional Abietadiene synthase from abies grandis
    Journal of Biological Chemistry, 2012
    Co-Authors: Ke Zhou, Francis M Mann, Richard B Honzatko, Reuben J Peters

    Abstract:

    Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg2+ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.

  • Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants.
    Phytochemistry, 2009
    Co-Authors: Ke Zhou, Reuben J Peters

    Abstract:

    Abstract Terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium, that are bound by a canonical DDXXD motif, as well as a putative second, seemingly less well conserved and understood (N/D)DXX(S/T)XXXE motif. Given the role of the Ser/Thr side chain hydroxyl group in ligating one of the three catalytically requisite divalent metal ions and the loss of catalytic activity upon substitution with Ala, it is surprising that Gly is frequently found in this ‘middle’ position of the putative second divalent metal binding motif in plant TPS. Herein we report mutational investigation of this discrepancy in a model plant diterpene cyclase, Abietadiene synthase from Abies grandis (AgAS). Substitution of the corresponding Thr in AgAS with Ser or Gly decreased catalytic activity much less than substitution with Ala. We speculate that the ability of Gly to partially restore activity relative to Ala substitution for Ser/Thr stems from the associated reduction in steric volume enabling a water molecule to substitute for the hydroxyl group from Ser/Thr, potentially in a divalent metal ion coordination sphere. In any case, our results are consistent with the observed conservation pattern for this putative second divalent metal ion binding motif in plant TPS.