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Rodney Croteau - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of monoterpene cyclization: stereochemistry of the transformation of noncyclizable substrate analogs by recombinant (−)-limonene synthase, (+)-bornyl diphosphate synthase, and (−)-pinene synthase
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Wilfried Schwab, David C Williams, Rodney CroteauAbstract:All monoterpene cyclases investigated to date are capable of overcoming the topological impediment to direct cyclization of the universal, acyclic C10 intermediate of isoprenoid biosynthesis geranyl diphosphate. Although strong suggestive evidence has been accumulated for the intermediary linalyl diphosphate in cyclase catalysis, all previous efforts to directly observe this product at the mandatory isomerization step have failed. (-)-4S-Limonene synthase from spearmint (Mentha spicata), (+)-bornyl diphosphate synthase from sage (Salvia officinalis), and (-)-pinene synthase from grand fir (Abies grandis) have been expressed in Escherichia coli and the recombinant enzymes have been isolated and purified. These enzymes were examined with the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and 2,3-methanogeranyl diphosphate to gain insight into the normally cryptic isomerization step of the reaction sequence. The analogs were catalytically active, affording acyclic olefins and alcohols as products. Chiral phase gas chromatography and mass spectrometry analysis provided evidence that the normal cyclization of geranyl diphosphate by (-)-4S-limonene synthase and by (-)-pinene synthase proceeds via preliminary isomerization to the bound tertiary intermediate 3S-linalyl diphosphate, whereas the cyclization catalyzed by (+)-bornyl diphosphate synthase proceeds via the intermediate 3R-linalyl diphosphate.
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geranyl diphosphate synthase from abies grandis cdna isolation functional expression and characterization
Archives of Biochemistry and Biophysics, 2002Co-Authors: Charles Burke, Rodney CroteauAbstract:Abstract Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir ( Abies grandis ) cDNA library. When expressed in Escherichia coli , three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k cat of 1.8 s −1 . Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.
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interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate
Journal of Biological Chemistry, 2002Co-Authors: Charles Burke, Rodney CroteauAbstract:Abstract Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C5 units, of the respective C10, C15, and C20 precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56–75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C10 chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.
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Mechanism of monoterpene cyclization: stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase.
Archives of biochemistry and biophysics, 2001Co-Authors: Wilfried Schwab, David C Williams, Edward M. Davis, Rodney CroteauAbstract:The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimately cyclizes to the various monoterpene skeletons. Previous experimental approaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and racemic methanogeranyl diphosphate, in attempts to dissect the cryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthases and by the inability to resolve chiral monoterpene products at the microscale. These approaches were revisited using three recombinant monoterpene synthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene olefin synthases, (−)-limonene synthase from spearmint and (−)-pinene synthase from grand fir, yielded essentially only achiral, olefin products (corresponding to the respective analogs and homologs of myrcene, trans-ocimene and cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogeranyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipated analog and homolog of the tertiary intermediate linalyl diphosphate (i.e., 6,7-dihydrolinalyl diphosphate and homolinalyl diphosphate, respectively). In the case of recombinant (+)-bornyl diphosphate synthase from common sage, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinalool and homolinalool also comprised significant components of the respective product mixtures, indicating greater access of water to the active site of this enzyme compared to the olefin synthases; again, no direct evidence for the production of 6,7-dihydrolinalyl diphosphate or homolinalyl diphosphate was obtained. Resolution of the terpenol products of (+)-bornyl diphosphate synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homolinalool from (2R,3S)-methanogeranyl diphosphate. The opposite stereochemistries of these products indicates water trapping from opposite faces of the corresponding tertiary carbocationic intermediates of the respective reactions, a phenomenon that appears to result from the binding conformations of these substrate analogs. Although these experiments failed to provide direct evidence for the tertiary intermediate of the tightly coupled isomerization-cyclization sequence, they did reveal a mechanistic difference between the olefin synthases and bornyl diphosphate synthase involving access of water as a participant in the reaction.
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stereochemical disposition of the geminal dimethyl groups in the enzymatic cyclization of geranyl diphosphate to bornyl diphosphate by recombinant bornyl diphosphate synthase from salvia officinalis
Tetrahedron, 2001Co-Authors: Mitchell L Wise, Hyung-jung Pyun, Greg Helms, Bryce Assink, Robert M Coates, Rodney CroteauAbstract:Abstract Regiospecifically deuterated geranyl diphosphate, in concert with NMR spectrometry, was employed to demonstrate that the trans -methyl group (C8) of geranyl diphosphate becomes the C9 carbon of (+)-bornyl diphosphate (geminal methyl syn to the diphosphate moiety) and that the cis -methyl group (C9) becomes the C8 (geminal methyl anti to the diphosphate). The syntheses of the relevant substrates and products, with accompanying spectrometric data are provided.
Kyozo Ogura - One of the best experts on this subject based on the ideXlab platform.
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An Artificial Substrate for the Thermostable Farnesyl Diphosphate Synthase from Bacillus stearothermophilus.
Chemistry Letters, 1997Co-Authors: Masahiko Nagaki, Yuji Maki, Kanako Shimizu, Kyozo OguraAbstract:Desmethyl homologs of dimethylallyl diphosphate and of isopentenyl diphosphate were examined for the reactivity as substrates for the thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus. E-But-2-enyl diphosphate was not accepted as a substrate at all, but but-3-enyl diphosphate reacted with dimethylallyl- and geranyl diphosphates to give norgeranyl- and norfarnesyl diphosphates, respectively, which lack methyl groups at the 3-positions.
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Purification and properties of geranylgeranyl-diphosphate synthase from bovine brain.
Journal of Biological Chemistry, 1994Co-Authors: Hiroshi Sagami, Yuiko Morita, Kyozo OguraAbstract:Abstract Geranylgeranyl-diphosphate synthase was purified to homogeneity from bovine brain in a one-step procedure employing an affinity column. For the construction of the affinity column, a farnesyl diphosphate analog, O-(6-amino-1-hexyl)-P-farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affi-Gel 10 via the amino group. The native enzyme appeared to be a homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pI for the enzyme was 6.2. The Km values for dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were estimated to be 33, 0.80, and 0.74 microM, respectively. The Km value for isopentenyl diphosphate in the reaction with isopentenyl diphosphate and farnesyl diphosphate was 2 microM. The reaction velocities for the formation of geranylgeranyl diphosphate from dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were in the ratio of 0.004:0.145:1. The intermediate farnesyl diphosphate was formed in the reaction with geranyl diphosphate as an allylic primer. Geranylgeranyl diphosphate acted as a competitive inhibitor against farnesyl diphosphate with an approximate Ki value of 1.2 microM in the condensation reaction of farnesyl diphosphate with isopentenyl diphosphate. Farnesyl-diphosphate synthase catalyzing the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate was also purified to homogeneity from the same organ by similar affinity chromatography using a geranyl diphosphate analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate, as a ligand. This enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that geranylgeranyl diphosphate, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from dimethylallyl diphosphate and isopentenyl diphosphate by the action of farnesyl-diphosphate synthase catalyzing the reaction of C5-->C15, followed by the action of geranylgeranyl-diphosphate synthase catalyzing a single reaction of C15-->C20, and that geranylgeranyl diphosphate can down-regulate its own synthesis through the inhibition of the geranylgeranyldiphosphate synthase action.
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Geranylgeranyl Diphosphate Synthase Catalyzing the Single Condensation between Isopentenyl Diphosphate and Farnesyl Diphosphate
Journal of biochemistry, 1993Co-Authors: Hiroshi Sagami, Tatsumi Korenaga, Kyozo OguraAbstract:Geranylgeranyl diphosphate synthase was purified 191-fold from bovine brain by Mono Q column chromatography followed by preparative isoelectric focusing electrophoresis and Superose 12 gel filtration. The synthase had a pI value at 6.0, and it was made free of farnesyl diphosphate synthase, the pI of which was 5.1. The partially purified enzyme catalyzed the formation of geranylgeranyl diphosphate from isopentenyl diphosphate and farnesyl diphosphate with the Km values for isopentenyl diphosphate and farnesyl diphosphate being 14 and 0.8 microM, respectively. Dimethylallyl diphosphate and geranyl diphosphate were poor substrates with velocities of only 0.003 and 0.03, respectively, relative to that of farnesyl diphosphate. These results indicate that geranylgeranyl diphosphate synthase catalyzes a single condensation between isopentenyl diphosphate and farnesyl diphosphate and that farnesyl diphosphate is the common intermediate at the branch point for the synthesis of geranylgeranylated proteins as well as cholesterol, ubiquinone, dolichol, and farnesylated proteins. The enzyme required Mg2+ or Mn2+ for maximum activity. Octylglucoside showed a stimulatory effect on the enzyme activity.
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Biosynthesis of prenyl diphosphates by cell-free extracts from mammalian tissues.
Journal of biochemistry, 1993Co-Authors: Hiroshi Sagami, Tatsumi Korenaga, Akira Kurisaki, Kyozo OguraAbstract:When assayed by the conventional method for prenyltransferase using a combination of [1-14C]isopentenyl and geranyl diphosphates, 100,000 x g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C]isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H]mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H]mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.
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Studies on geranylgeranyl diphosphate synthase from rat liver: specific inhibition by 3-azageranylgeranyl diphosphate.
Archives of biochemistry and biophysics, 1992Co-Authors: Hiroshi Sagami, Hyung-jung Pyun, Kyozo Ogura, Tatsumi Korenaga, Arthur Steiger, Robert M CoatesAbstract:Geranylgeranyl diphosphate synthase from rat liver was separated from farnesyl diphosphate synthase, the most abundant and widely occurring prenyltransferase, by DEAE-Toyopearl column chromatography. The enzyme catalyzed the formation of E,E,E-geranylgeranyl diphosphate (V) from isopentenyl diphosphate (II) and dimethylallyl diphosphate (I), geranyl diphosphate (III), or farnesyl diphosphate (IV) with relative velocities of 0.09:0.15:1. 3-Azageranylgeranyl diphosphate (VII), designed as a transition-state analog for the geranylgeranyl diphosphate synthase reaction, was synthesized and found to act as a specific inhibitor for this synthase, but not for farnesyl diphosphate synthase. Diphosphate V and its Z,E,E-isomer (VI) also inhibited geranylgeranyl diphosphate synthase, but the effect was not as striking as that of the aza analog VII. Specific inhibition of geranylgeranyl diphosphate synthase by VII was also observed in experiments with 100,000g supernatants of rat brain and liver homogenates which contained isopentenyl diphosphate isomerase and prenyltransferases including farnesyl diphosphate synthase as well as geranylgeranyl diphosphate synthase. For farnesyl:protein transferase from rat brain, however, the aza compound did not show a stronger inhibitory effect than E,E,E-geranylgeranyl diphosphate.
Hiroshi Sagami - One of the best experts on this subject based on the ideXlab platform.
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Specificity of geranylgeranyl diphosphate synthase for homoallylic substrate analogs
Journal of Molecular Catalysis B: Enzymatic, 2015Co-Authors: Norimasa Ohya, Hiroshi Sagami, Takumi Ichijo, Hana Sato, Takeshi Nakamura, Saki Yokota, Masahiko NagakiAbstract:Abstract The goal of this study was to determine the substrate specificity of Homo sapiens geranylgeranyl diphosphate synthase (GGPPase) for analogs of isopentenyl diphosphate (IPP) to facilitate the application to organic synthesis techniques to the study of prenyl chain elongation enzymes. For this purpose, we used the IPP analogs 2a–d, which contain different alkyl side-chains at the 3-position, as substrates of the condensation reaction with the allylic substrate geranyl diphosphate (GPP) that is catalyzed by GGPPase. GGPPase catalyzed the reaction of GPP with 3-desmethylisopentenyl diphosphate (but-3-enyl diphosphate) to yield 3-desmethylfarnesyl diphosphate (12.1%), as well as the reaction of GPP with 3-ethylbut-3-enyl diphosphate or 3-propylbut-3-enyl diphosphate to yield 3-ethylfarnesyl diphosphate (46.9%) or 3-propylfarnesyl diphosphate (22.6%), respectively. However, a reaction product was not detected when 3-butylbut-3-enyl diphosphate was used as substrate.
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Substrate specificities of E- and Z-farnesyl diphosphate synthases with substrate analogs
Journal of Molecular Catalysis B: Enzymatic, 2012Co-Authors: Masahiko Nagaki, Norimasa Ohya, Takumi Ichijo, Tohru Musashi, Jun Kawakami, Rikiya Kobashi, Yusuke Yagihashi, Takeshi Gotoh, Hiroshi SagamiAbstract:Abstract Prenyltransferases catalyzes the basic isoprenoid chain elongation to produce prenyl diphosphates, which led to upward of 30,000 diverse isoprenoids as steroids, carotenoids, natural rubbers, and prenyl proteins. Here, we determined the reactivities of E- and Z-farnesyl diphosphate synthases (E- and Z-FPP synthases) isolated from Bacillus stearothermophilus and Thermobifida fusca, respectively. For this purpose we use the synthetic substrate analogs, 8-tetrahydropyran-2-yloxy-, 8-hydroxy- and 8-acetoxygeranyl diphosphates. Z-FPP synthase catalyzed the reaction between 8-hydroxygeranyl diphosphate (HOGPP) and isopentenyl diphosphate (IPP), which produced (2Z)-12-hydroxyfarnesyl diphosphate (yield: 16.7%) and (2Z, 6Z)-16-hydroxygeranylgeranyl diphosphate (yield: 6.6%). Neither E- nor Z-farnesyl diphosphate synthases detectably catalyzed reactions between 8-tetrahydropyran-2-yloxygeranyl diphosphate (8-THPOGPP) and IPP. However, a mutated E-FPP synthase (Y81S), did catalyze this reaction, producing 12-tetrahydropyran-2-yloxyfarnesyl diphosphate (12-THPOFPP) with a yield of 12.3%. Wild-type E-FPP synthase catalyzed the reaction of 8-acetoxygeranyl diphosphate (8-AcOGPP) with IPP, which produced 12-acetoxyfarnesyl diphosphate (12-AcOFPP) (yield, 21.8%). Mutant E-FPP synthase catalyzed the reaction between 8-AcOGPP with IPP, producing 12-AcOFPP and 16-acetoxygeranylgeranyl diphosphate (16-AcOGGPP) with respective yields of 55.3% and 1.7%. We believe our results contribute to a better understanding of the catalytic properties of these key enzymes and illustrate their use in the stereo-specific syntheses of compounds that may have significant biotechnological and medical applications.
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Purification and properties of geranylgeranyl-diphosphate synthase from bovine brain.
Journal of Biological Chemistry, 1994Co-Authors: Hiroshi Sagami, Yuiko Morita, Kyozo OguraAbstract:Abstract Geranylgeranyl-diphosphate synthase was purified to homogeneity from bovine brain in a one-step procedure employing an affinity column. For the construction of the affinity column, a farnesyl diphosphate analog, O-(6-amino-1-hexyl)-P-farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affi-Gel 10 via the amino group. The native enzyme appeared to be a homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pI for the enzyme was 6.2. The Km values for dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were estimated to be 33, 0.80, and 0.74 microM, respectively. The Km value for isopentenyl diphosphate in the reaction with isopentenyl diphosphate and farnesyl diphosphate was 2 microM. The reaction velocities for the formation of geranylgeranyl diphosphate from dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were in the ratio of 0.004:0.145:1. The intermediate farnesyl diphosphate was formed in the reaction with geranyl diphosphate as an allylic primer. Geranylgeranyl diphosphate acted as a competitive inhibitor against farnesyl diphosphate with an approximate Ki value of 1.2 microM in the condensation reaction of farnesyl diphosphate with isopentenyl diphosphate. Farnesyl-diphosphate synthase catalyzing the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate was also purified to homogeneity from the same organ by similar affinity chromatography using a geranyl diphosphate analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate, as a ligand. This enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that geranylgeranyl diphosphate, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from dimethylallyl diphosphate and isopentenyl diphosphate by the action of farnesyl-diphosphate synthase catalyzing the reaction of C5-->C15, followed by the action of geranylgeranyl-diphosphate synthase catalyzing a single reaction of C15-->C20, and that geranylgeranyl diphosphate can down-regulate its own synthesis through the inhibition of the geranylgeranyldiphosphate synthase action.
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Geranylgeranyl Diphosphate Synthase Catalyzing the Single Condensation between Isopentenyl Diphosphate and Farnesyl Diphosphate
Journal of biochemistry, 1993Co-Authors: Hiroshi Sagami, Tatsumi Korenaga, Kyozo OguraAbstract:Geranylgeranyl diphosphate synthase was purified 191-fold from bovine brain by Mono Q column chromatography followed by preparative isoelectric focusing electrophoresis and Superose 12 gel filtration. The synthase had a pI value at 6.0, and it was made free of farnesyl diphosphate synthase, the pI of which was 5.1. The partially purified enzyme catalyzed the formation of geranylgeranyl diphosphate from isopentenyl diphosphate and farnesyl diphosphate with the Km values for isopentenyl diphosphate and farnesyl diphosphate being 14 and 0.8 microM, respectively. Dimethylallyl diphosphate and geranyl diphosphate were poor substrates with velocities of only 0.003 and 0.03, respectively, relative to that of farnesyl diphosphate. These results indicate that geranylgeranyl diphosphate synthase catalyzes a single condensation between isopentenyl diphosphate and farnesyl diphosphate and that farnesyl diphosphate is the common intermediate at the branch point for the synthesis of geranylgeranylated proteins as well as cholesterol, ubiquinone, dolichol, and farnesylated proteins. The enzyme required Mg2+ or Mn2+ for maximum activity. Octylglucoside showed a stimulatory effect on the enzyme activity.
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Biosynthesis of prenyl diphosphates by cell-free extracts from mammalian tissues.
Journal of biochemistry, 1993Co-Authors: Hiroshi Sagami, Tatsumi Korenaga, Akira Kurisaki, Kyozo OguraAbstract:When assayed by the conventional method for prenyltransferase using a combination of [1-14C]isopentenyl and geranyl diphosphates, 100,000 x g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C]isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H]mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H]mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.
C. D. Poulter - One of the best experts on this subject based on the ideXlab platform.
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isoprenyl diphosphate synthases protein sequence comparisons a phylogenetic tree and predictions of secondary structure
Protein Science, 2008Co-Authors: A Chen, P A Kroon, C. D. PoulterAbstract:Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain‐elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate‐rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of α‐helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families. Copyright
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Purification and characterization of farnesyl diphosphate/geranylgeranyl diphosphate synthase. A thermostable bifunctional enzyme from Methanobacterium thermoautotrophicum.
The Journal of biological chemistry, 1993Co-Authors: Anjun Chen, C. D. PoulterAbstract:Abstract Farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase, a bifunctional enzyme that synthesizes C15 and C20 isoprenoid diphosphates from isopentenyl diphosphate and dimethylallyl diphosphate, was purified to homogeneity from the archaebacterium Methanobacterium thermoautotrophicum. The only activities detected from synthesis of FPP and GGPP copurified through (NH4)2SO4 precipitation and four chromatographic steps. The pure enzyme was a 79-kDa homodimer that catalyzed the sequential addition of isopentenyl diphosphate to dimethylallyl diphosphate, geranyl diphosphate, and FPP by a non-processive mechanism which allowed substantial amounts of FPP to accumulate during turnover, creating a pool for further elongation to GGPP or for synthesis of squalene. The bifunctional enzyme required Mg2+ or Mn2+ and was optimally active at 65 degrees C. Catalysis of chain elongation in M. thermoautotrophicum differs from related reactions in eubacteria and eukaryotes, where distinct FPP synthases and GGPP synthases are found.
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purification and characterization of farnesyl diphosphate geranylgeranyl diphosphate synthase a thermostable bifunctional enzyme from methanobacterium thermoautotrophicum
Journal of Biological Chemistry, 1993Co-Authors: Anjun Chen, C. D. PoulterAbstract:Abstract Farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase, a bifunctional enzyme that synthesizes C15 and C20 isoprenoid diphosphates from isopentenyl diphosphate and dimethylallyl diphosphate, was purified to homogeneity from the archaebacterium Methanobacterium thermoautotrophicum. The only activities detected from synthesis of FPP and GGPP copurified through (NH4)2SO4 precipitation and four chromatographic steps. The pure enzyme was a 79-kDa homodimer that catalyzed the sequential addition of isopentenyl diphosphate to dimethylallyl diphosphate, geranyl diphosphate, and FPP by a non-processive mechanism which allowed substantial amounts of FPP to accumulate during turnover, creating a pool for further elongation to GGPP or for synthesis of squalene. The bifunctional enzyme required Mg2+ or Mn2+ and was optimally active at 65 degrees C. Catalysis of chain elongation in M. thermoautotrophicum differs from related reactions in eubacteria and eukaryotes, where distinct FPP synthases and GGPP synthases are found.
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Analysis and purification of phosphorylated isoprenoids by reversed-phase HPLC.
Analytical Biochemistry, 1993Co-Authors: Donglu Zhang, C. D. PoulterAbstract:Procedures were developed for analysis and purification of phosphorylated isoprenoids by reversed-phase HPLC. Dimethylallyl diphosphate (C5), geranyl diphosphate (C10), farnesyl diphosphate (C15), geranylgeranyl diphosphate (C20), and farnesylgeranyl diphosphate (C25) were cleanly separated on a C18 column by gradient elution with acetonitrile and 25 mM NH4HCO3. Reversed-phase HPLC was used to directly analyze extracts for phosphorylated isoprenoids and to follow the progress of enzyme-catalyzed reactions without having to hydrolyze the phosphate esters before analysis. Examples are provided for geranylgeranyl diphosphate synthase, geranylgeranylglyceryl phosphate synthase, and squalene synthase. The separations were run on preparative scales to purify products from the enzymatic reactions for mass spectrometry and to purify synthetic substrates.
Masahiko Nagaki - One of the best experts on this subject based on the ideXlab platform.
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Specificity of geranylgeranyl diphosphate synthase for homoallylic substrate analogs
Journal of Molecular Catalysis B: Enzymatic, 2015Co-Authors: Norimasa Ohya, Hiroshi Sagami, Takumi Ichijo, Hana Sato, Takeshi Nakamura, Saki Yokota, Masahiko NagakiAbstract:Abstract The goal of this study was to determine the substrate specificity of Homo sapiens geranylgeranyl diphosphate synthase (GGPPase) for analogs of isopentenyl diphosphate (IPP) to facilitate the application to organic synthesis techniques to the study of prenyl chain elongation enzymes. For this purpose, we used the IPP analogs 2a–d, which contain different alkyl side-chains at the 3-position, as substrates of the condensation reaction with the allylic substrate geranyl diphosphate (GPP) that is catalyzed by GGPPase. GGPPase catalyzed the reaction of GPP with 3-desmethylisopentenyl diphosphate (but-3-enyl diphosphate) to yield 3-desmethylfarnesyl diphosphate (12.1%), as well as the reaction of GPP with 3-ethylbut-3-enyl diphosphate or 3-propylbut-3-enyl diphosphate to yield 3-ethylfarnesyl diphosphate (46.9%) or 3-propylfarnesyl diphosphate (22.6%), respectively. However, a reaction product was not detected when 3-butylbut-3-enyl diphosphate was used as substrate.
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Substrate specificities of E- and Z-farnesyl diphosphate synthases with substrate analogs
Journal of Molecular Catalysis B: Enzymatic, 2012Co-Authors: Masahiko Nagaki, Norimasa Ohya, Takumi Ichijo, Tohru Musashi, Jun Kawakami, Rikiya Kobashi, Yusuke Yagihashi, Takeshi Gotoh, Hiroshi SagamiAbstract:Abstract Prenyltransferases catalyzes the basic isoprenoid chain elongation to produce prenyl diphosphates, which led to upward of 30,000 diverse isoprenoids as steroids, carotenoids, natural rubbers, and prenyl proteins. Here, we determined the reactivities of E- and Z-farnesyl diphosphate synthases (E- and Z-FPP synthases) isolated from Bacillus stearothermophilus and Thermobifida fusca, respectively. For this purpose we use the synthetic substrate analogs, 8-tetrahydropyran-2-yloxy-, 8-hydroxy- and 8-acetoxygeranyl diphosphates. Z-FPP synthase catalyzed the reaction between 8-hydroxygeranyl diphosphate (HOGPP) and isopentenyl diphosphate (IPP), which produced (2Z)-12-hydroxyfarnesyl diphosphate (yield: 16.7%) and (2Z, 6Z)-16-hydroxygeranylgeranyl diphosphate (yield: 6.6%). Neither E- nor Z-farnesyl diphosphate synthases detectably catalyzed reactions between 8-tetrahydropyran-2-yloxygeranyl diphosphate (8-THPOGPP) and IPP. However, a mutated E-FPP synthase (Y81S), did catalyze this reaction, producing 12-tetrahydropyran-2-yloxyfarnesyl diphosphate (12-THPOFPP) with a yield of 12.3%. Wild-type E-FPP synthase catalyzed the reaction of 8-acetoxygeranyl diphosphate (8-AcOGPP) with IPP, which produced 12-acetoxyfarnesyl diphosphate (12-AcOFPP) (yield, 21.8%). Mutant E-FPP synthase catalyzed the reaction between 8-AcOGPP with IPP, producing 12-AcOFPP and 16-acetoxygeranylgeranyl diphosphate (16-AcOGGPP) with respective yields of 55.3% and 1.7%. We believe our results contribute to a better understanding of the catalytic properties of these key enzymes and illustrate their use in the stereo-specific syntheses of compounds that may have significant biotechnological and medical applications.
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Substrate specificities of farnesyl diphosphate synthases with respect to cyclic substrate homologs
Transactions of the Materials Research Society of Japan, 2010Co-Authors: Tohru Musashi, Norimasa Ohya, Saki Yokota, Jun Kawakami, Hiroshi Kanno, Masahiko NagakiAbstract:We investigated substrate specificities of farnesyl diphosphate synthases (FPSs) derived from porcine liver and Bacillus stearothermophilus by examining the reactivity of cyclopentylideneethyl diphosphate with several 3-alkyl homologs of isopentenyl diphosphate. Reaction of cyclopentylideneethyl diphosphate with isopentenyl diphosphate using porcine liver or bacterial enzyme gave 10-cyclopentyliden-3,7-dimethyldeca-2,6-dinenyl diphosphate as a double condensation product, with relative yields of 40.9% for the porcine liver enzyme and 15.9% for the bacterial enzyme. Reaction of cyclohexlideneethyl diphosphate with 3-ethylbut-3-enyl diphosphate using the bacterial enzyme gave 10-cyclohexliden-3,7-diethyldeca-2,6-dinenyl diphosphate (yield: 24.6%).
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Substrate specificities of farnesyl diphosphate synthases of Bacillus stearothermophilus and porcine liver with allylic substrate homologs having vinyl or ethynyl group
Journal of Molecular Catalysis B-enzymatic, 2009Co-Authors: Masahiko Nagaki, Tohru Musashi, Yuji Hirano, Hidenori Tanaka, Junji Ichita, Yuji MakiAbstract:Abstract To investigate substrate specificities of farnesyl diphosphate synthases from porcine liver and Bacillus stearothermophilus , we have examined the reactivities of vinyldimethylallyl and ethynyldimethylallyl diphosphates as allylic substrate homologs. The reaction of vinyldimethylallyl diphosphate with isopentenyl diphosphate by farnesyl diphosphate synthase of porcine liver gave vinylgeranyl and vinylfarnesyl diphosphates, which shows that the reaction stopped at the single or double condensation of isopentenyl diphosphate, respectively. However, the similar reaction by the use of wild-type farnesyl diphosphate synthase of B. stearothermophilus gave vinylfarnesyl diphosphate, exclusively. On the other hand, the reaction of Z -ethynyldimethylallyl diphosphates with isopentenyl diphosphate by the use of wild-type of farnesyl diphosphate synthase of B. stearothermophilus gave Z -ethynylfarnesyl diphosphate, as the sole product. Moreover, a mutated farnesyl diphosphate synthase (Y81D) reaction of Z -ethynyldimethylallyl diphosphates with isopentenyl diphosphate gave three kinds of products: ethynylgeranyl, ethynylfarnesyl, and ethynylgeranylgeranyl diphosphates. Using wild-type of farnesyl diphosphate synthase of B. stearothermophilus , the reaction of E -ethynyldimethylallyl diphosphate with isopentenyl diphosphate gave only E -ethynylfarnesyl diphosphate as double condensation product.
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Substrate specificities of wild and mutated farnesyl diphosphate synthases: Reactivity of allylic substrate homologs having hydrophilic groups at ω-position
Journal of Molecular Catalysis B: Enzymatic, 2009Co-Authors: Masahiko Nagaki, Minori Nakada, Tohru Musashi, Jun Kawakami, Takae Endo, Yuji MakiAbstract:Abstract To investigate substrate specificities of wild and mutated types of farnesyl diphosphate synthases from Bacillus stearothermophilus , we have examined the reactivities of methoxymethoxydimethylallyl- and propoxygeranyl diphosphates as allylic substrate homologs. The wild type farnesyl diphosphate synthase reaction of methoxymethoxydimethylallyl and propoxygeranyl diphosphates with isopentenyl diphosphate gave methoxymethoxygeranyl and propoxyfarnesyl diphosphates which stopped at the first stage of the condensation. Using a mutated farnesyl diphosphate synthase (Y81D FPS), the reaction of methoxymethoxydimethylallyl diphosphate with isopentenyl diphosphate gave only methoxymethoxyfarnesyl diphosphate as single condensation product. Moreover, both of mutated farnesyl diphosphate synthase reaction with propoxygeranyl diphosphate of isopentenyl diphosphate gave propoxyfarnesyl- and propoxygeranylgeranyl diphosphate.