Search

Chloroplastsisolated in organic solvents (“non-aqueous chloroplasts”)incorporate [2-14C]-mevalonate into geranylgeraniol and phytoene. No incorporation into phytol or into other carotenoids was achieved, even in mixtures of non-aqueous chloroplasts with chloroplasts prepared in aqueous media. The incorporation of geranylgeranyl pyrophosphate into phytoene by the non-aqueous chloroplast preparation has been demonstrated, and the stereochemistry of hydrogen loss in phytoene synthesis by non-aqueous chloroplasts has been determined.

The esterifying alcohols of protochlorophyll a and 4-vinyl-(4-desethyl)-protochlorophyll a (purified as the respective pheophytins) from pumpkin seeds were examined by gas chromatography-mass spectrometry. The results of the analysis suggested that pumpkin seed protochlorophyll a is esterified with all possible C20 isoprenoid alcohols between and including geranylgeraniol and phytol, phytol comprising 90% or more of the mixture of esterifying alcohols, and that the 4-vinyl-(4-desethyl)-protochlorophyll a is esterified with farnesol and all possible C20 isoprenoid alcohols between and including geranylgeranoid and phytanol, phytol comprising 50% or more of the mixture of esterifying alcohols. The 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of older mature pumpkin seeds was found to be richer in esterifying alcohols corresponding to isoprenoid precursors of phytol then was the 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of younger mature seeds. Other isoprenoid alcohols may have been present in very minor quantities in the mixtures of esterifying alcohols from the pumpkin seed protochlorophylls but were not looked for in this study. These results are discussed in terms of a biosynthetic accumulation of 4-vinyl-(4-desethyl)-protochlorophyll a in pumpkin inner seed-coat tissue.

The protochlorophyllide ester isolated from dark grown barley leaves was shown to contain geranylgeraniol as esterifying alcohol. No phytylester was found. The qualitative analyses were performed with combined gas chromatography-mass spec-trometry. Chromatographic separation and spectrofluorometric determination of the protochlorophyll and chlorophyll pigments before and after irradiation of the dark grown leaves with light flashes at 2°C showed that part of the protochlorophyllide ester was photoconverted to chlorophyll a.

Geranylgeraniol, (—)-kaurene, (—)-kaurenol, (—)-kaurenal and (—)-kaurenoic acid were tentatively identified as products synthesized from mevalonic acid-2 14 C in cell-free reaction mixtures containing 10,000 g supernatant of extracts of cotyledons of immature pea ( Pisum sativum L. cv. Alaska) seeds. The identity of (—)-kaurenol was confirmed by co-crystallization of the radioactive product with authentic kaurenol to constant specific radioactivity. Repeated efforts to observe cell-free metabolism of exogenous (—)-kaurene in a variety of enzyme extracts consistently resulted in failure. Only when (—)-kaurene was synthesized in situ from mevalonic acid in reaction mixtures containing 10,000 g supernatant from cotyledon extracts could the metabolism of (—)-kaurene be observed. Thus, the procedure employed most extensively was to allow (—)-kaurene to be formed from mevalonic acid-2 14 C for 60 min, then to inhibit its further synthesis by the addition of AMO-1618, and finally to assay the carbon monoxide-sensitive oxidation of (—)-kaurene which occurred during a subsequent 60-min incubation. By this procedure it was demonstrated that the oxidation of (—)-kaurene which was formed in situ was enzymic, or at least heat labile; markedly sensitive to inhibition by carbon monoxide; and greater in extracts prepared with insoluble polyvinyl-pyrrolidone (PVP) than in extracts prepared without PVP. Possible reasons for the failure of exogenous (—)-kaurene to be oxidized are discussed.

A description is given of the total synthesis of the isoprenold alcohols linalool, geraniol, nerol, nerolidol, farnesol, geranyllinalool, geranylgeraniol, and phytol and also of the intermediate products in this synthesis.

The plant growth retardants (2-chloroethyl)-trimethylammonium chloride (CCC) and 2'-isopropyl-4'-(trimethylammoniumchloride)-5'-methylphenyl-piperidine-1-carboxylate (AMO-1618) inhibit gibberellic-acid biosynthesis inFusarium moniliforme at the cyclisation of geranylgeraniol to (-)-kaurene, causing an accumulation of geranylgeraniol. The two inhibitors have no effect on the biosynthesis of ergosterol inF. moniliforme or sitosterol in barley seedlings.

Kaurene, squalene and phytoene were identified as products from mevalonate-2- 14 C in a soluble system from young pea fruits. All three hydrocarbons were obtained when the incubation mixture contained ATP, Mg 2+ , Mn 2+ and pyridine nucleotides. Only squalene required pyridine nucleotides and only phytoene required Mn 2+ . The omission of pyridine nucleotides therefore leads to a specific exclusion of squalene synthesis and the omission of Mn 2+ to a specific exclusion of phytoene synthesis. In addition, the formation of squalene was inhibited over a certain range of Mn 2+ -concentrations, while the formation of phytoene was stimulated. Therefore the ratio of these two products was strongly dependent on the Mn 2+ -concentration. The formation of kaurene was optimal at a lower pH than that for the formation of the other products. Only kaurene synthesis was inhibited by the plant growth inhibitor AMO 1618. A mixed plastid and mitochondrial fraction greatly increased the formation of squalene, farnesol and geranylgeraniol but not the formation of phytoene and kaurene. One or more unidentified acids were also formed in the system.

14 C- trans -Geranylgeranyl pyrophosphate has been identified as a product formed from 2- 14 C-mevalonate in a soluble enzyme fraction of an homogenate of endosperm tissue of immature Echinocystis macrocarpa seed. A method based on published procedures has been developed for the purification of this material from incubation mixtures. This depends on (1) collidine (2-methyl-5-ethylpyridine) extraction of the incubation mixture, (2) transfer of the pyrophosphate ester to 0.01 m sodium bicarbonate solution from a collidine-ether mixture, and (3) chromatography of the bicarbonate extract on a DBAE-Sephadex A-25 column developed with an ammonium bicarbonate gradient. trans -Geranylgeranyl pyrophosphate was identified as the major radioactive component on the basis of the following characteristics: (1) geranylgeraniol and geranyllinalool were identified as the principal radioactive products of acid hydrolysis; (2) geranylgeraniol was identified as the radioactive product of hydrolysis with bacterial alkaline phosphatase; and (3) two moles of phosphate per mole of suspected geranylgeranyl pyrophosphate were released after hydrolysis in 1 m HCl at 100 ° for 10 minutes. The biosynthesized trans -geranylgeranyl pyrophosphate was converted in yields as high as 90% to the cyclic diterpene (−)-kaurene in the presence of enzyme preparations from E. macrocarpa . Inclusion of the plant growth retardant Amo 1618 (2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine-1-carboxylate) in the incubation medium increased the level and duration of accumulation of trans -geranylgeranyl pyrophosphate. The evidence suggests that this is a normal intermediate in the formation of (−)-kaurene and geranylgeraniol in the endosperm tissue.

Farnesol, geranylgeraniol, dolichols and ubiquinones were the main radioactive components of the unsaponifiable lipid recovered from Phytophthora cactorum grown in aerated cultures containing [2-14C]mevalonate. The 14C recovered in each of these components was in the approximate proportion 2:4:3:5. When the culture was not aerated no radioactive ubiquinone was recovered. Most of the 14C recovered in the dolichols was found in dolichol−15 (37%), with decreasing amounts in dolichol−14 (30%) and −13 (14%) and only a little (5%) in dolichol−16, whereas the major components, by weight, of the mixture (13μg/g of damp-dry tissue) were dolichol−14, −15 and −16 in the approximate proportion of 1:3:1. Radioautography of appropriate chromatograms indicated the presence also of traces of radioactivity in dolichol-9, −10, −11, −12 and −17. Most (80%) of the 14C recovered in the ubiquinones was associated with ubiquinone-9, the rest being in ubiquinone-8. Most (80%) of the weight of ubiquinones (19μg/g of damp-dry tissue) was also ubiquinone-9. The identification of these compounds was by chromatographic methods and, for the ubiquinones and dolichols, was confirmed by mass spectrometry. In addition, the incorporation of 4R- and/or 4S-3H from [4-3H]-mevalonates showed the expected stereochemistry of biosynthesis, namely that farnesol, geranylgeraniol and ubiquinones were biogenetically all trans and the dolichols each contained three biogenetically trans isoprene residues, the remaining residues being biogenetically cis. The distribution of 14C in the components of the whole lipid of the fungus was consistent with 97% of both the farnesol and geranylgeraniol being present as the fatty acid ester. The corresponding value for dolichols was 37%. The observation by other workers, that this fungus does not form either squalene or sterol, was confirmed.

Gibberellic acid (GA 3 ) and 13-deoxy-gibberellic acid (GA 7 ) were identified in extracts of germinating barley as their 14 C-methyl esters. The maximal level of GA 3 was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2- 14 C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA 3 could not be detected. Cell-free embryo extracts from germinating barley converted 2- 14 C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while 14 C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent -kaurene.

Biosynthesis of the isoprenols, geraniol, farnesol, nerolidol, and geranylgeraniol, from [2- 14 C]mevalonic acid is described in the fly, Sarcophaga bullata . Each was identified by thin-layer and gas chromatographic, infra-red spectroscopic, and mass spectrometric analyses. These isoprenols underwent structural rearrangement when acid hydrolysis was used to isolate them from their pyrophosphates. Most of the farnesyl pyrophosphate was converted to nerolidol, while only a fraction of the geranyl pyrophosphate was changed to its isomers. Since farnesol is the major isoprenol product of the in vivo incubation, we conclude that this insect is capable of isoprenoid biosynthesis at least to this point, and that the block in the cholesterol biosynthetic pathway lies beyond.

Methyl chlorophyllide a , but not chlorophyllide a , was esterified with phytol (or geranylgeraniol) in the presence of chlorophyllase from Chlorella protothecoides . No detectable esterifying activity was found when the pyrophosphate derivatives of phytol and geranylgeraniol were used.

Incubation of (2-14C)-mevalonic acid with 3000 × g adult rat brain supernate in the presence of Tween 80 or Triton X-100 produced a marked shift in the distribution of radioactivity among squalene, steryl ester and free sterol when compared to controls. Although the conversion of labeled mevalonate to cholesterol was only slightly influenced, the detergents markedly affected the turnover of squalene and methyl sterol precursors. The major neutral isoprenoid material formed had the general properties of geranylgeraniol on TLC and radioactivity-monitored-GLC. Phospholipase A from Vipera russelli, at an optimal tissue to phospholipase ratio, resulted in a 25% increase in mevalonate incorporation into total neutral isoprenoids. An increase in radioactivity of almost 25% was also seen in the free sterol fraction. Phospholipase A from bee venom did not show this type of increase. No labeled geranylgeraniol-type material was found on incubation with either of the phospholipases.

Excised shoots and cell-free extracts prepared from Wedgwood iris (Iris hollandica Hoog. "Wedgwood") shoots metabolized (14)C-labeled mevalonic acid (MVA). By using cell-free extracts, the (14)C from MVA-1-(14)C was recovered as (14)CO(2), while that from MVA-2-(14)C was recovered as neutral terpenes, acid-hydrolyzable terpenes, or (14)CO(2). Also, under optimal incubation conditions, 12.8 nanomoles R-MVA-2-(14)C was incorporated into neutral terpenes per milligram fresh weight per hour. In contrast, excised shoots incorporated only 0.58 nanomoles R-MVA-2-(14)C per mg fresh weight per hour. Labeled products identified from the cell-free system were squalene, farnesol, geranylgeraniol, and compounds that are converted to farnesol and geranylgeraniol after alkaline hydrolysis. Squalene and a 4,4-dimethylsterol were identified as products from excised shoots but not the terpene alcohols or the alkaline-hydrolyzable compounds.

Publisher Summary Terpenes are formed by the combination of isoprenoid units to form compounds, such as geraniol (C10), farnesol (C15), geranylgeraniol (C20), squalene (C80), and others of a similar kind, and can be derived from these precursors by accepted cyclization and, in certain cases, rearrangement mechanisms. This chapter describes the biosynthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Hemiterpenes are classified as free hemiterpenes, phenolic hemiterpenes, alkaloids with hemiterpene residues, isopentenyl adenosine, and related compounds. Similarly, monoterpenes are grouped into acyclic monoterpenes, monocyclic monoterpenes, bicyclic monoterpenes, phenolic monoterpenoids, alkaloids with monoterpene residues, and atypical monoterpenes. The main groups of sesquiterpenes are: acyclic compounds such as farnesol; monocyclic systems such as bisabolol; and bicyclic systems, which are subdivided into four main groups, the cadalene type [e.g., (+)-γ-cadinene], the eudesmol type (e.g. α-eudesmol), the iresin type, and the spiro-4,5-decane type (e.g.β-vetivone). Diterpenes are studied as monocyclic diterpenes, bicyclic diterpenes, tricyclic diterpenes, tetracyclic diterpenes, and diterpene derivatives. All triterpenes are formed by cyclization of all-transsqualene in various chair-boat conformational sequences. Biosynthesis of tetraterpenoids, pentaterpenoids, and polyterpenoids are also discussed. Rubber is a polyterpenoid with a molecular weight that varies from 20,000 to several millions according to source and location in the plant.

By using stereospecifically double-labelled radioactive mevalonates it was shown that betulaprenols-6 to -9, found in the woody tissue of Betula verrucosa, each contain three biogenetically trans-isoprene residues and that the remaining residues are biogenetically cis. The results obtained with these radioactive mevalonates also indicated that the activity of isopentenyl pyrophosphate isomerase is low relative to the activity of prenyltransferase in this woody tissue. The incorporation of radioactive farnesyl pyrophosphate and radioactive geranylnerol and the lack of incorporation of radioactive geranylgeraniol into betulaprenols-6 to -9 demonstrated that they are formed by the cis-additions of isoprene residues to all-trans-farnesyl pyrophosphate.

Publisher Summary This chapter discusses the enzymatic synthesis of phytoene in tomato. An enzyme system present in the chromoplasts of tomato fruit converts either isopentenyl pyrophosphate or farnesyl plus isopentenyl pyrophosphates to phytoene. Isopentenyl pyrophosphate is required for the conversion of farnesyl pyrophosphate to phytoene. Concomitantly with the synthesis of phytoene from isopentenyl pyrophosphate, acid-labile compounds accumulate that can be cleaved with bacterial alkaline phosphatase to yield, primarily, geranylgeraniol. Hence, it is assumed that geranylgeranyl pyrophosphate is an intermediate and that two molecules of this compound condense to form phytoene. The chapter discusses assay for isopentenyl-PP isomerase and subsequent condensing enzyme activities and preparation of tomato enzyme system. The chapter concludes with a discussion on the properties of enzyme system.

A cell-free system consisting of a soluble fraction and plastid fragments from pea shoots incorporates 2-(14)C-mevalonate very actively into farnesol, squalene, geranylgeraniol, and other isoprenoids of different carbon-chain lengths. The products have different cofactor requirements, which makes it possible to channel the pathway into different products by varying the incubation mixture.

The incorporation of [2− 14 C,(5 R )-5− 3 H 1 ]MVA* and [2− 14 C,5− 3 H 2 ]MVA into geranylgeraniol and phytoene by a preparation of ‘non-aqueous’ bean leaf chloroplasts has been studied. In the formation of phytoene from two molecules of geranylgeranyl pyrophosphate, the loss of hydrogen is stereospecific, the hydrogen atom lost from C-1 of each molecule of geranylgeranyl pyrophosphate being that which was originally the pro-S hydrogen atom from C-5 of mevalonate. All the pro-R hydrogen atoms from C-5 of mevalonate are retained. These results with a cell-free system confirm and extend the observations made in previous work with tomato slices.

The ability of fourteen marine invertebrates to utilize [14C]mevalonate for the biosynthesis of isoprenoid compounds was investigated. Several of the animals, in particular crustaceans, bivalve molluscs, a coelenterate and a sponge, were unable to synthesize squalene and sterols, whereas gastropod molluscs, echinoderms, an annelid and a sponge could. Regardless of sterol-synthesizing ability the animals (with the exception of a sponge) always made dolichol and ubiquinone, and thus a specific block in squalene and sterol synthesis was indicated in some animals. Radioactivity accumulated in relatively large amounts in farnesol and geranylgeraniol in those animals incapable of making sterols.

Geranylgeraniol and (–)-labda-8,13-dien-15-ol pyrophosphate have been shown to act as precursors of (–)-kaurene, the kaurenolides, and gibberellic acid. The labelling pattern from 4-(R)-[4-3H]- and 2-[3H2]-mevalonic acid in these tetracyclic diterpenes has been determined and this evidence is used to exclude (–)-primara-7,8- and -8,9-dienes from the biosynthesis. (–)Pimara-8(14)-diene has been shown to be specifically incorporated into the kaurenolides and gibberellic acid. The stereochemistry of hydroxylation of ring A of the gibberellins has been shown to proceed with retention of configuration. The loss of the angular C-20 atom in the formation of the C-19 gibberellins does not involve the decarboxylation of a 4-, 4b-, or 10a-unsaturated acid.