Enzymes
UniProtKB help_outline | 6 proteins |
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- Name help_outline (+)-copalyl diphosphate Identifier CHEBI:58635 Charge -3 Formula C20H33O7P2 InChIKeyhelp_outline JCAIWDXKLCEQEO-ATPOGHATSA-K SMILEShelp_outline [H][C@@]12CCC(=C)[C@H](CC\C(C)=C\COP([O-])(=O)OP([O-])([O-])=O)[C@@]1(C)CCCC2(C)C 2D coordinates Mol file for the small molecule Search links Involved in 14 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline abieta-7,13-diene Identifier CHEBI:30232 (Beilstein: 2504776; CAS: 35241-40-8) help_outline Charge 0 Formula C20H32 InChIKeyhelp_outline BBPXZLJCPUPNGH-CMKODMSKSA-N SMILEShelp_outline [H][C@]12CCC(=CC1=CC[C@@]1([H])C(C)(C)CCC[C@]21C)C(C)C 2D coordinates Mol file for the small molecule Search links Involved in 2 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline diphosphate Identifier CHEBI:33019 (Beilstein: 185088) help_outline Charge -3 Formula HO7P2 InChIKeyhelp_outline XPPKVPWEQAFLFU-UHFFFAOYSA-K SMILEShelp_outline OP([O-])(=O)OP([O-])([O-])=O 2D coordinates Mol file for the small molecule Search links Involved in 1,085 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Cross-references
RHEA:13873 | RHEA:13874 | RHEA:13875 | RHEA:13876 | |
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Reaction direction help_outline | undefined | left-to-right | right-to-left | bidirectional |
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Publications
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Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production.
Zerbe P., Chiang A., Yuen M., Hamberger B., Hamberger B., Draper J.A., Britton R., Bohlmann J.
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, ... >> More
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 bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. << Less
J. Biol. Chem. 287:12121-12131(2012) [PubMed] [EuropePMC]
This publication is cited by 5 other entries.
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Evolution of conifer diterpene synthases: diterpene resin acid biosynthesis in lodgepole pine and jack pine involves monofunctional and bifunctional diterpene synthases.
Hall D.E., Zerbe P., Jancsik S., Quesada A.L., Dullat H., Madilao L.L., Yuen M., Bohlmann J.
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 ... >> More
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. << Less
Plant Physiol. 161:600-616(2013) [PubMed] [EuropePMC]
This publication is cited by 5 other entries.
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The primary diterpene synthase products of Picea abies levopimaradiene/abietadiene synthase (PaLAS) are epimers of a thermally unstable diterpenol.
Keeling C.I., Madilao L.L., Zerbe P., Dullat H.K., Bohlmann J.
The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies; PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. Howeve ... >> More
The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies; PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. However, variability in the assay products observed by GC-MS of this and orthologous conifer diterpene synthases over the past 15 years suggested that these diterpenes may not be the initial enzyme assay products but are rather the products of dehydration of an unstable alcohol. We have identified epimers of the thermally unstable allylic tertiary alcohol 13-hydroxy-8(14)-abietene as the products of PaLAS. The identification of these compounds, not previously described in conifers, as the initial products of PaLAS has considerable implications for our understanding of the complexity of the biosynthetic pathway of the structurally diverse diterpene resin acids of conifer defense. << Less
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Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the 'pseudomature' recombinant enzyme.
Peters R.J., Flory J.E., Jetter R., Ravn M.M., Lee H.J., Coates R.M., Croteau R.B.
The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abiet ... >> More
The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps. << Less
Biochemistry 39:15592-15602(2000) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Bifunctional abietadiene synthase: free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites.
Peters R.J., Ravn M.M., Coates R.M., Croteau R.B.
Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranyl ... >> More
Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed. << Less
J. Am. Chem. Soc. 123:8974-8978(2001) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase.
Wilderman P.R., Peters R.J.
Terpene synthases often catalyze complex cyclization reactions that typically represent the committed step in particular biosynthetic pathways, leading to great interest in their enzymatic mechanisms. We have recently demonstrated that substitution of a specific Ile with Thr was sufficient to "sho ... >> More
Terpene synthases often catalyze complex cyclization reactions that typically represent the committed step in particular biosynthetic pathways, leading to great interest in their enzymatic mechanisms. We have recently demonstrated that substitution of a specific Ile with Thr was sufficient to "short circuit" the complex cyclization reaction normally catalyzed by ent-kaurene synthases to instead produce ent-pimaradiene. Here we report that the complex cyclization/rearrangement reaction catalyzed by abietadiene synthase can be similarly cut short to produce pimaradienes by an analogous Ser for Ala change, albeit with a slight shift in active site location to accommodate the difference in substrate stereochemistry. This result has mechanistic implications for enzymatic catalysis of abietadiene cyclization, and terpene synthases more broadly. Furthermore, these defined single residue switches may be useful in engineering product outcome in diterpene synthases more generally. << Less
J. Am. Chem. Soc. 129:15736-15737(2007) [PubMed] [EuropePMC]
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Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate.
Peters R.J., Croteau R.B.
Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinc ... >> More
Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, geranylgeranyl diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the diphosphate moiety to assist in positioning the substrate for catalysis. A putative active site for the protonation-initiated cyclization was defined by modeling abietadiene synthase and locating the DXDD motif previously shown to be involved in this reaction. A number of charged and aromatic residues, which are highly conserved in mechanistically related diterpene cyclases, line the putative active site. Alanine substitutions were made for each of these residues, as were asparagine and glutamate substitutions for the aspartates of the DXDD motif. Kinetic evaluation confirmed the involvement of most of the targeted residues in the reaction, and analysis of mutational effects on the pH-activity profile and affinity for a transition state analogue suggested specific roles for several of these residues in catalyzing the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. A functional role was also suggested for the cryptic insertional element found in abietadiene synthase and other diterpene synthases that carry out similar protonation-initiated cyclizations. << Less
Biochemistry 41:1836-1842(2002) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Abietadiene synthase catalysis: mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement.
Peters R.J., Croteau R.B.
Abietadiene synthase catalyzes the committed step in resin acid biosynthesis, forming a mixture of abietadiene double-bond isomers by two sequential, mechanistically distinct cyclizations at separate active sites. The first reaction, protonation-initiated cyclization, converts the universal diterp ... >> More
Abietadiene synthase catalyzes the committed step in resin acid biosynthesis, forming a mixture of abietadiene double-bond isomers by two sequential, mechanistically distinct cyclizations at separate active sites. The first reaction, protonation-initiated cyclization, converts the universal diterpene precursor geranylgeranyl diphosphate to the stable bicyclic intermediate copalyl diphosphate. In the second, magnesium ion-dependent reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone and is coupled, by intramolecular proton transfer within a transient pimarenyl intermediate, to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane structure. Alternative deprotonations of the terminal abietenyl carbocation provide a mixture of abietadiene, levopimaradiene, and neoabietadiene, and this product profile varies as a function of pH. Mutational analysis of amino acids at the active site of a modeled structure has identified residues critical for catalysis, as well as several that play roles in specifying product formation, apparently by ligation of a magnesium ion cofactor. These results strongly suggest that choice between alternatives for deprotonation of the abietenyl intermediate depends more on the positioning effects of the carbocation-diphosphate anion reaction partners than on the pKa of multiple participating bases. In one extreme case, mutant N765A is unable to mediate the intramolecular proton transfer and aborts the reaction, without catalyzing 1,2-methyl migration, to produce only sandaracopimaradiene, thereby providing supporting evidence for the corresponding stereochemistry of the cryptic pimarenyl intermediate of the reaction pathway. << Less
Proc. Natl. Acad. Sci. U.S.A. 99:580-584(2002) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Mechanism of abietadiene synthase catalysis: stereochemistry and stabilization of the cryptic pimarenyl carbocation intermediates.
Ravn M.M., Peters R.J., Coates R.M., Croteau R.
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 ... >> More
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 8 alpha-hydroxy-17-nor CPP (19a) to 17-normanoyl oxide (20a) and the higher inhibitory potency of the norpimarenylamine 26a (K(i) = 0.1 nM) both suggest pimarenyl intermediates having the 13 beta methyl configuration and 8,14-double bond corresponding to sandaracopimaradiene (5a). The 2000-fold stimulation of inhibition by 26a in the presence of inorganic pyrophosphate indicates an important role for carbocation/OPP anion stabilization of the secondary sandaracopimaren-15-yl(+) ion. The failure of 8 beta-hydroxy-17-nor CPP (19b) to undergo enzymatic cyclization was taken as evidence that 9 is bound with a "coplanar" side chain conformation and that the S(N)' cyclization occurs on the 17 alpha face. The routing of the sandarcopimara-15-en-8-yl carbocation toward various diterpenes in biogenetic schemes is attributed to differing conformations of ring C and/or orientations of the C13 vinyl group in the active sites of the corresponding diterpene cyclases. << Less
J Am Chem Soc 124:6998-7006(2002) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.