Kaurene Synthetase Research Articles

The Biosynthesis of Abscisic Acid in a Cell-free System from Embryos of Hordeum vulgare

Summary Cell-free homogenates of imbibed embryos of Hordeum vulgare which actively synthesized terpenyl pyrophosphates also transformed mevalonic acid and isopentenyl pyrophosphate into the sesquiterpenoid plant growth hormone, abscisic acid. Abscisic acid biosynthesis was stimulated by the addition of the squalene 2,3-oxide cyclase and kaurene synthetase inhibitor, AMO1618 and a «cold-pool trap» of (±) abscisic acid but was inhibited by cytokinins. This is the first demonstration of abscisic acid biosynthesis by a cell-free system derived from non-vegetative higher plant tissues which could prove useful for elucidating the biosynthetic route to this compound in plants.

Diallate inhibition of gibberellin biosynthesis in sorghum coleoptiles

Diallate [S-(2,3-dichloroallyl)diisopropylthiocarbamate] incorporated into sand significantly inhibited sorghum ( Sorghum bicolor (L.) Moench cv Funks G 522DR) growth of 14-day-old seed lings. Inhibition was competitively reversed by exogenous giberellic acid (GA 3) (0.1 and 10 ppmw). Diallate inhibited gibberellin (GA) precursor biosynthesis in a cell-free enzyme preparation from unruptured, etiolated sorghum coleoptiles. Diallate (10 μ M) inhibited kaurene oxidation 40% with a 2.7 × increase in kauren-ol and a 50% decrease in kaurenoic acid. The GA biosynthesis inhibition correlates with symptom phenology and field use application concentrations. Geranylgeranyl pyrophosphate accumulated 5 × at 0.1 μ M diallate concentrations but concomitant kaurene concentration decreases did not occur. At 10 μ M diallate, kaurene synthetase was inhibited 33%.

Distribution of ent-kaurene synthetase in Helianthus annuus and Marah macrocarpus

Kaurene synthetases catalyse the biosynthesis of ent-kaurene, a precursor of the gibberellins. In 4-day-old dark- or light-grown Helianthus annuus seedlings, the cotyledons contained over 90% of the synthetase activity. The low enzyme activity in the seedling hypocotyls and roots is not a consequence of inhibitory factors in these tissues. The cotyledons not only have the highest kaurene synthetase activity, but also have the highest inhibitory activity. The differences in kaurene synthetase activities in the different tissues cannot be explained on the basis of the levels of inhibitor(s) in the extracts. The mature perennial root of Marah macrocarpus has very low kaurene synthetase activity, in contrast to the liquid endosperm of immature seeds of the same plant which is a rich source of the enzyme.

Ent-kaurene synthesis in chloroplasts from higher plants

Lysed chloroplasts from several higher plants synthesized ent-kaurene from copalyl pyrophosphate but not from geranylgeranyl pyrophosphate. The copalyl pyrophosphate transforming activity (so-called B activity of kaurene synthetase) was relatively stable in plastid lysates from Pisum sativum but remarkably unstable in similar preparations of Hordeum vulgare. The bulk of the B activity of kaurene synthetase appeared to reside in the stroma of plastids from P. sativum but required the presence of plastid membranes for maximum activity.

Kaurene Synthetase Activity in Helianthus annuus L.

In previous studies, the conversion of geranylgeranyl pyrophosphate to ent-kaurene (kaurene synthetase AB activity) could not be detected readily in crude extracts of sunflower (Helianthus annuus L.) seedlings (Shen-Miller, West 1982 Plant Physiol 69: 637-641). These investigations also revealed the presence of inhibitors for Marah macrocarpus kaurene synthetase AB activity in crude extracts of sunflower seedlings. It has now been found that crude extracts prepared from intact sunflower seedlings stored in liquid N(2) for several days have greatly enhanced AB activity in comparison with frozen, but not stored, controls. The levels of activity for the conversion of copalyl pyrophosphate to ent-kaurene (kaurene synthetase B activity) are affected only slightly by storage of intact seedlings in liquid N(2). Extracts from intact seedlings that had been stored in liquid N(2) also showed less inhibitory activity for Marah macrocarpus endosperm kaurene synthetase AB activity.

Ent-Kaurene Biosynthesis in Extracts of Helianthus annuus L. Seedlings

Kaurene synthetase B activity (conversion of copalyl pyrophosphate to ent-kaurene) is readily detectable in crude cell-free extracts of 3- to 4-day old dark-grown sunflower (Helianthus annuus cv. Mammoth) seedlings, whereas little or no kaurene synthetase AB activity (conversion of geranylgeranyl pyrophosphate to ent-kaurene) can be found in these extracts under comparable assay conditions. A low amount of AB activity is evident only if an extensively dialyzed extract is used in low concentrations as the enzyme source. One factor which may contribute to the low apparent levels of AB activity is the presence of inhibitory factors in the crude sunflower extract since these extracts can be shown to act as a potent inhibitor of Marah macrocarpus endosperm kaurene synthetase AB activity. Heat treatment (100 degrees C) or dialysis of the sunflower extract reduces the amount of its inhibitory activity. Also, it was observed that low concentrations of extensively dialyzed sunflower extracts act to stimulate M. macrocarpus AB activity. There is no evidence for the presence of an inhibitory factor for M. macrocarpus kaurene synthetase B activity in sunflower extracts. However, there does appear to be present in the crude preparation of sunflower extract a dialyzable factor(s) that impedes its own B activity. There is little information to date on the nature of these inhibitory and stimulatory factors for kaurene synthetase activity or their possible roles in physiological regulation. The possible presence of such factors should be considered, however, when attempting to evaluate kaurene synthetase activities in extracts of vegetative plants.

The effects of gamma-radiation and 2-chloroethyl trimethyl ammonium chloride on biosynthesis of ent(-)kaurene in germinating wheat

The effect of gamma-radiation (doses ranging from 0.2 to 2kGy) on in vitro synthesis of ent(-)kaurene was investigated using a partially purified ent(-)kaurene synthetase preparation from germinating wheat endosperms. Incorporation of 2- 14C-mevalonic acid (MVA) into ent(-)kaurene reaches a maximum at 56 hr of germination in all the samples, but the rate decreases with increasing radiation dose. Similarly, biosynthesis of gibberellin (GA) in seeds, imbided with 2- 13C-MVA, is reduced by about 50%, whereas its biological activity is only 2% of control at 2 kGy. This may be attributed to higher sensitivity of the biosynthesis of GA 3, the most biologically active GA derivative in wheat. This reduction in GA synthesis is accompanied by a concomitant accumulation of intermediate precursors, GA 4 and GA 7, in irradiated wheat seedlings. These results indicate that radiation treatment reduces the efficiency of interconversion of a less active GA to a highly active one. Pre-treatment of seeds with 2-chloroethyl trimethyl ammonium chloride (CCC) inhibits ent(-)kaurene synthesis in vitro, but only at high concentrations. Similarly, CCC inhibits GA synthesis in vivo and accentuates the radiation induced damage in wheat seedlings.

Properties of Kaurene Synthetase from Marah macrocarpus Endosperm: Evidence for the Participation of Separate but Interacting Enzymes

Ent-kaurene is synthesized from geranylgeranyl pyrophosphate in a two step sequence catalyzed by kaurene synthetase; the first step (A activity) involves the conversion of geranylgeranyl pyrophosphate into the intermediate ent-trans labda-8(17), 13-dien-15-yl pyrophosphate (copalyl pyrophosphate) which is further cyclized to ent-kaurene in the second step (B activity). The resolution of enzyme fractions which catalyze each step independent of the other has been accomplished for the first time by means of QAE Sephadex A-50 chromatography and polyacrylamide gel electrophoresis of kaurene synthetase preparations from endosperm tissue of immature seed of Marah macrocarpus. Molecular weights for the A and B enzymes were each estimated as approximately 82,000 by means of gel filtration chromatography and sedimentation velocity determinations.Experiments in which [(14)C]geranylgeranyl pyrophosphate and [(3)H]copalyl pyrophosphate were incubated simultaneously with kaurene synthetase preparations demonstrated that copalyl pyrophosphate derived from [(14)C]geranylgeranyl pyrophosphate is more readily converted to kaurene than is exogenously added [(3)H]copalyl pyrophosphate. This implies that copalyl pyrophosphate derived from the catalytic site of the A enzyme is preferentially channelled to the B enzyme catalytic site for conversion to ent-kaurene, rather than freely equilibrating with a pool of copalyl pyrophosphate in the medium. Experiments in which the rates of the overall AB and independent B activities of kaurene synthetase preparations were measured as a function of total protein concentration further suggest that overall AB activity is catalyzed by an AB enzyme complex which is in equilibrium with free A and B enzymes. A model is proposed for M. macrocarpus kaurene synthetase in which separate but interacting A and B enzymes must associate for the efficient production of ent-kaurene from geranylgeranyl pyrophosphate.

Inhibition of kaurene synthetase from castor bean seedling and the germination of tomato, wheat and castor bean seeds by di-ethylene glycol disulfide

Di-ethylene glycol disulfide (ESSE) inhibits the enzyme kaurene synthetase derived from castor bean ( Ricinus communis L.) seedlings. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and had little or no effect on the further cylization of copalyl pyrophosphate to polycyclic diterpene hydrocarbons (activity B). The inhibition of activity A of kaurene synthetase has a non-competitive reversible pattern ( K i = 10 μM) with regard to geranylgeranyl pyrophosphate. ESSE inhibits the germination of tomato ( Solanum lycopersicum L.) wheat ( Triticum aestivum) and castor bean ( Ricinus communis L.) seeds. The inhibition is reversible and normal germination was restored by washing the seeds with H 2O.GA 3 failed to promote germination of ESSE-treated tomato seeds.

Isolation of a Kaurene Synthetase Inhibitor from Castor Bean Seedlings and Cell Suspension Cultures

Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures and seedlings of castor bean. Both cell-free extracts contain an inhibitor of kaurene synthetase. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and has little or no effect on the further cyclization of copalyl pyrophosphate to ent-kaurene (activity B) in both castor bean and Fusarium moniliforme cell-free enzyme preparations. In castor bean cell suspension cultures, the inhibitor diffuses out of the cells to the growth medium. The inhibitor is stable to 100 C heat treatment for 10 minutes and exposure to pH values of 2.0 or 13.0, and it diffuses through a dialysis bag (10(4)-dalton cutoff). Gel filtration chromatography of the inhibitor on a calibrated Bio-Gel P-10 column indicated a molecular weight of 7,500. Kinetic studies indicate that the inhibition of activity of A of kaurene synthetase is noncompetitive and reversible.

Properties of Kaurene Synthetase from Marah macrocarpus

The kaurene synthetase from immature seeds of Marah macrocarpus (Greene) Greene was partially purified from cell-free homogenates of endosperm by a combination of QAE-Sephadex A-25 chromatography and hydroxyapatite chromatography and freed of contaminating phosphatase activity. The two catalytic activities associated with kaurene synthetase, the cyclization of geranylgeranyl-pyrophosphate to copalyl-pyrophosphate (activity A) and the cyclization of copalyl-pyrophosphate to ent-kaurene (activity B), were not even partially resolved from one another during these procedures. Both activities had identical elution profiles from a calibrated Sepharose 4B column corresponding to a molecular weight less than that of ovalbumin (45,000).The A and B activities had pH optima of 7.3 and 6.9, respectively. Both activities required millimolar concentrations of the following divalent cations in the order: Mg(2+) > Mn(2+) > Co(2+). Activities A and B were both sensitive to inhibition by Hg(2+), Cu(2+), p-hydroxymercuribenzoate, and N-ethylmaleimide, but activity B was much more sensitive than activity A. The average value of Km' (apparent Km in the absence of substrate inhibition) for geranylgeranyl-pyrophosphate was 1.6 mum. Values of 0.5 and 0.6 mum were obtained for Km' and Km, respectively, for copalyl-pyrophosphate. The Vm' values for the two activities were similar: 12 and 9 pmol/minute.mug protein for activities A and B, respectively.N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and N,N-dimethylaminoethyl-2,2-diphenylphentyl ether (SKF-3301A), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D), tributyl-2,4-dichlorobenzylammonium chloride (Phosfon S), 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo-1618), 2-(N,N-dimethyl-N-heptylammonium bromide)-p-methan-1-ol (Q-58), and 2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol (Q-64), at concentrations from 1 to 5 mum, were effective inhibitors of kaurene synthetase activity A. Acetylcholine chloride and 2-chloroethyl-trimethylammonium chloride were effective inhibitors of activity A only at concentrations of 5 mm or greater. Abscisic acid, indole-3-acetate, gibberellin A(1), gibberellin A(3), a mixture of gibberellins A(4) and A(7), gibberellin A(13), and N,N-dimethylaminosuccinamic acid (B995) were not inhibitory at any of the levels tested. None of these compounds was an effective inhibitor of activity B at concentrations less than 0.5 mm.

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Farnesyl pyrophosphate-[ 14C] and geranylgeranyl pyrophosphate-[ 14C] were biosynthesized from mevalonic acid-[2- 14C] by cell-free enzyme extracts of pea ( Pisum sativum) cotyledons containing MgCl 2, MnCl 2, ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[ 14C] served as an efficient substrate for ent-kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent-kaurene in very low yields. Ent-kaurene production from mevalonic acid-[2- 14C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.

Kaurene synthetase from plastids of developing plant tissues

Summary Kaurene synthesis from copalyl pyrophosphate is present in the plastid fraction from all three sources of developing plant tissue studied. Isolated plastids from Echinocystis macrocarpa endosperm have kaurene synthetic capacity from either geranylgeranyl pyrophosphate or copalyl pyrophosphate. However, organelle preparations from etiolated pea shoot tips or developing castor bean endosperm have either no or a very limited ability to synthesize kaurene from geranylgeranyl pyrophosphate. These results suggest that the first committed step in gibberellin biosynthesis, geranylgeranyl pyrophosphate conversion to copalyl pyrophosphate, may be limited under certain stages of plant development.

Purification and Properties of Kaurene Synthetase from Fusarium moniliforme

Abstract Kaurene synthetase has been purified approximately 170-fold from cell-free extracts of the fungus Fusarium moniliforme Sheld. The two catalytic activities associated with kaurene synthetase, namely the cyclization of trans-geranylgeranyl-PP to copalyl-PP (activitiy A) and the cyclization of copalyl-PP to (-)-kaurene (activity B), copurify and have not been resolved by any procedures tested. Both activities are associated with a major band of protein after the purified enzyme is subjected to polyacrylamide gel electrophoresis in a pH 8 system. Gel electrophoresis at pH 10.2 of the purified enzyme or in the presence of sodium dodecyl sulfate results in the appearance of many protein bands, none of which retains either catalytic activity. The molecular weight of both the crude and purified enzyme was estimated to be 4.3 to 4.9 x 105 from gel filtration chromatography and sucrose density gradient centrifugation. Prenyl transferase, squalene synthetase, squalene 2,3-oxide cyclase, and kaurene oxidase activities were absent from the purified enzyme. These general properties suggest that kaurene synthetase may be associated with a complex of proteins, although the nature of other polypeptides in the complex is unknown. Activities A and B showed pH optima of 7.5 and 6.9, respectively. Both activities required divalent metal ions with Mg2+ the most active. Ni2+ at 0.1 mm to 1.0 mm supported activity A but not activity B; advantage was taken of this in the biosynthesis of substrate quantities of copalol in crude extracts of the fungus. In the presence of EDTA, dithiothreitol stimulates activity B but not activity A. Several reagents which react with sulfhydryl groups were effective in blocking both activities, but to different extents; in general, activity B is more sensitive to these agents than activity A. The Km for geranylgeranyl-PP in activity A was determined to be approximately 0.7 µm. The Km for copalyl-PP in activity B was approximately 1.0 µm, although this determination was complicated by substrate inhibition at higher substrate concentrations. Several potential inhibitors of both activities were tested. 2'-Isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo 1618) and tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D) are representative of plant growth retardants which strongly inhibit activity A (Ki for Amo 1618 = 2.3 x 10-7 m), but not activity B. These substances are noncompetitive inhibitors with respect to geranylgeranyl-PP. 2-(N,N-Dimethyl-N-octylammonium bromide)-p-menthan-1-ol (Q-64) and N,N-dimethylaminoethyl 2,2-diphenylpentyl ether (SKF 3301A) are representative of substances which are reasonably effective inhibitors of both activities. Deoxycholate was an inhibitor of activity B, whereas PPi and gibberellic acid were not appreciably active as inhibitors of either activity. Kinetic studies in the presence of both substrates by dual isotope techniques have indicated a preferential utilization in the conversion to kaurene of the copalyl moiety generated from geranylgeranyl-PP in comparison with the pool of added copalyl-PP. These studies also demonstrated an inhibition of activity A by copalyl-PP which is competitive with respect to geranylgeranyl-PP.