Mevalonate-activating enzymes in callus culture cells from Nepeta cataria

The 30000 g supernatants from cell-free extracts of Nepeta cataria leaf tissue and leaf callus tissue have mevalonic acid kinase, mevalonic acid phosphate kinase and mevalonic acid pyrophosphate decarboxylase activities. The callus tissue cell-free extract produced mevalonic acid pyrophosphate and isopentenyl pyrophosphate; however, very little mevalonic acid phosphate was observed. The leaf cell-free extracts incubated with [¹⁴C]-mevalonic acid produced higher amounts of mevalonic acid phosphate. When both the leaf cell-free extract and the callus cell-free extract were incubated with [¹⁴C]-mevalonic acid in the presence of iodoacetamide, the ion exchange column elution profile was cleaner, which was confirmed by PC. Apparently the callus tissue 30000 g supernatant contains mevalonic acid phosphorylating enzymes even though there is no production of the methyl cyclopentane monoterpenes.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Carotene biosynthesis by a cell extract of Aspergillus giganteus mut alba

A cell extract prepared from lyophilized mycelia of light-grown cultures of Aspergillus giganteus mut alba converted [2-¹⁴C]mevalonic acid into phytoene, lycopene, β-carotene and squalene, but from similar preparations from dark grown cultures formed only squalene. The carotenogenic activities of the cell extracts varied with the age of the cultures. Phytoene synthetase was located in the cytosolic fraction, whereas the dehydrogenation and cyclisation steps were catalysed by membrane-bound enzymes. Dithiothreitol, ATP, Mn²⁺, Mg²⁺, NAD and NADP were essential for the formation of carotenes from mevalonic acid, whilst FAD was required for phytoene metabolism. Oxygen enhanced the conversion of phytoene into other carotenes.     

Comparative study of in vivo stigmasterol biosynthesis in Nicotiana tabacum and Hordeum vulgare

Six-day-old tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]sitosterol but neither plant was able to convert it into stigmasterol. However, a sterol metabolite was isolated from both species and the acetate derivative was slightly more polar, on AgNO₃—silica gel TLC, than stigmasteryl acetate. A similar metabolite was also obtained with [4-¹⁴C]cholesterol, indicating a general metabolic reaction of plants to exogenous sterols. Both species incorporated [2-¹⁴C]mevalonic acid into sitosterol and stigmasterol. We suggest that in vascular plants, whether monocotyledons or dicotyledons, the pathway of stigmasterol biosynthesis is not via sitosterol but through a common precursor which is derived from mevalonic acid.     

Stereochemical course of the methylation of the polygalacturonic acid carboxyl groups of pectin

The steric course of the methyl group transfer to polygalacturonic acid to form the methyl ester group in pectin was studied using S-adenosylmethionine (AdoMet) carrying a methyl group made chiral by labeling with ¹H, ²H, ³H, in an asymmetric arrangement. The incubation of the two diastereomers of this substrate with a particulate enzyme preparation obtained from Phaseolus aureus (mung bean) shoots gave the corresponding pectins. These were degraded in a series of stereochemically unambiguous reactions that converted the methoxy group into the methyl group of acetate, which was then analyzed for its configuration. The results indicate that the transfer of the methyl group from the sulfur of AdoMet to the oxygen of the carboxyl group proceeds with inversion of configuration of the methyl group.     

Metabolism of Mevalonic Acid in Vegetative and Induced Plants of Xanthium strumarium

The metabolism of mevalonic acid in Xanthium strumarium L. Chicago plants was studied to determine how mevalonate was metabolized and whether metabolism was related to induction of flowering. Leaves of vegetative, photoperiodically induced, and chemically inhibited cocklebur plants were supplied with [¹⁴C]mevalonic acid prior to or during a 16-hour inductive dark period. Vegetative, induced, and Tris(2-diethylaminoethyl)phosphate trihydrochloride-treated plants did not differ significantly in the amount of [¹⁴C]mevalonic acid they absorbed, nor in the distribution of radioactivity among the leaf blade (97%), petiole (2.3%), or shoot tip (0.7%). [¹⁴C]Mevalonic acid was rapidly metabolized and transported out of the leaves. Possible metabolites of mevalonate were mevalonic acid phosphates and sterols. No detectable ¹⁴C was found in gibberellins, carotenoids, or the phytol alcohol of chlorophyll. Chemically inhibited plants accumulated ¹⁴C compounds not found in vegetative or induced plants. When ethanol extracts of leaves, petioles, and buds were chromatographed, comparisons of chromatographic patterns did not show significant differences between vegetative and induced treatments.     

Biogenesis of cyclobuxine-D and cyclovirobuxine-D in Buxus sempervirens

Two major alkaloids from Buxus sempervirens, cyclovirobuxine-D and cyclobuxine-D, were found to be radioactively labelled following administration of mevalonic acid [2-¹⁴C,(4R)-4-³H₁] to freshly-harvested shoots. The ³H: ¹⁴C atomic ratio of 3:4 in cyclovirobuxine-D indicated a biosynthetic pathway from cycloartenol involving 3-ketone and 20-ketone intermediates. A ³H: ¹⁴C atomic ratio of ca 3:3 in cyclobuxine-D suggests that the 4α-methyl group of cycloartenol is lost in its formation, and this conforms with current theories of the sequence of C-4 demethylation of sterols.     

Biosynthesis of nimbolide from [2-14C,(4R)4-3H1] mevalonic acid lactone in the leaves of Azadirachta indica

Nimbolide was biosynthesized from [2-¹⁴C, (4R)4-³H₁]mevalonic acid lactone in the leaves of Azadirachta indica. The nimbolide had a ³H:¹⁴C ratio of 3:5 which gives support to the suggestion of the involvement of a triterpenoid intermediate with a double bond at the Δ⁸⁽⁹)-position in the biosynthesis of nimbolide.     

Biosynthesis of α-spinasterol from (2- 14C (4R)-4-3H1) mevalonic acid by Spinacea oleracea and Medicago sativa

Leaves of Spinacea oleracea and Medicago sativa were incubated with (2-¹⁴C, (4R)-4³H₁ mevalonic acid and the sterols isolated. Cycloartenol had a ³H: ¹⁴C atomic ratio of 6:6 whilst oxidation to cycloartenone resulted in a ratio of 5:6 showing that tritium was present in the 3α-position and that the cycloartenol was symmetrically labelled. Separation of the 4-demethyl sterols gave α-spinasterol and a mixture of stigmast-7-enol and 24-methylcholest-7-enol, which had ³H: ¹⁴C atomic ratios of 3:5. Ozonolysis of α-spinastery] acetate gave the terminal side chain fragment as 2-ethyl-3-methyl butanoic acid. The acid contained ¹⁴C but no tritium thus showing that the C-24 hydrogen of cycloartenol is lost during the alkylation reactions leading to the C-24 ethyl group of α-spinasterol.     

Biosynthesis of andrographolide in Andrographis paniculata

Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-¹³C]acetate, [2-¹³C]acetate and [1,6-¹³C₂]glucose. The peak enrichment of eight carbon atoms in the ¹³C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene.The contribution of the mevalonic acid pathway (MVA) is indicated by the observed ¹³C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm.     

Biosynthesis of fomannoxin in the root rotting pathogen Heterobasidion occidentale

Fomannoxin is a biologically active benzohydrofuran, which has been suggested to be involved in the pathogenicity of the root rotting fungus Heterobasidion annosum sensu lato. The biosynthesis of fomannoxin was investigated through an isotopic enrichment study utilizing [1-¹³C]glucose as metabolic tracer. ¹³C NMR spectroscopic analysis revealed the labeling pattern and showed that the isoprene building block originates from the mevalonic acid pathway, whereas the aromatic motif is formed via the shikimic acid route by elimination of pyruvate from chorismic acid. A natural product, 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde (1), was isolated and characterized, and was suggested to be a key intermediate in the biosynthesis of fomannoxin and related secondary metabolites previously identified from the H. annosum fungal species complex.     

Biosynthesis of the prenyl side chains of plastiquinone and related compounds in maize and barley shoots

The incorporation of ¹⁴C by etiolated maize and barley shoots exposed to light of ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into phylloquinone, plastoquinone, ubiquinone, α-tocopherolquinone and α-tocopherol was examined. In maize (the principal tissue studied) it was demonstrated that ¹⁴C from [2-¹⁴C]mevalonic acid is incorporated into phylloquinone, plastoquinone and ubiquinone. α-Tocopherol and α-tocopherolquinone, although undoubtedly labelled from this substrate, were not purified completely. As expected, ¹⁴C from ¹⁴CO₂ was incorporated into all components examined. Ozonolytic degradation studies showed that ¹⁴C from [2-¹⁴C]mevalonic acid was incorporated specifically into the prenyl side chains of plastoquinone and ubiquinone, and from this it was inferred that mevalonic acid can be regarded as the specific distal precursor to the prenyl portions of all terpenoid quinones occurring in plant tissues. From a comparison of the relative incorporation of ¹⁴C from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into the intra- and extra-chloroplastidic terpenoids evidence was obtained consistent with the tenet that the prenyl portions of the chloroplastidic quinones phylloquinone and plastoquinone, along with β-carotene, are biosynthesized within the confines of the chloroplast, the side chain of the extraplastidic ubiquinone and phytosterols being synthesized elsewhere within the cell. The results obtained for the incorporation of ¹⁴C from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into α-tocopherol and α-tocopherolquinone were not readily interpretable with regard to the site of synthesis of these compounds.     

Rapid feedback inhibition of endogenous cholic and chenodeoxycholic acid synthesis by exogenous chenodeoxycholic acid in man.

Chenodeoxycholic acid (300 mg + ¹⁴C) was administered orally to a bile fistula patient receiving a constant infusion of {³H}mevalonic acid. Suppression of endogenous cholic and chenodeoxycholic acid synthesis occurred within 2 to 4 hours and continued for the next 10 hours; synthesis returned to the baseline level after 18 hours. Incorporation of {³H}mevalonic acid into both bile acids was also greatly reduced during the first several hours after chenodeoxycholic acid, but almost recovered by 5 hours. The data suggest that multiple feedback sites are involved in the regulation of bile acid synthesis in man.     

Stereospecificity of biosynthesis of triterpene alcohols in Calendula officinalis flowers

Flowers of Calendula officinalis were incubated with mevalonic acid doubly labelled with ¹⁴C in position 2 and ³H in positions 2R, 2S, 4R or 5R,S and the [³H/¹⁴C] ratios determined in squalene and pentacylic mono- and dihydroxy-triterpene alcohols and also in some derivatives prepared from the triterpene alcohols. ³H atoms were located in positions 3, 12, 16, 21, 29, 30 of the ursane skeleton, positions 3, 12, 29, 30 of the lupane skeleton and positions 3, 11, 12, 18 of the oleanane skeleton. Stabilization of α- and β-Amyrins, ω-taraxasterol and lupeol occurs with the elimination of a proton from positions 12, 21 and 29 (or 30) respectively. In addition, during hydroxylation of triterpene monols to the corresponding diols a proton is substituted by the hydroxyl group.     

In vitro production of metabolism-enhancing phytoecdysteroids from <Emphasis Type="Italic">Ajuga turkestanica</Emphasis>

In order to develop a sustainable source of metabolism-enhancing phytoecdysteroids, cell suspension and hairy root cultures were established from shoot cultures of wild-harvested Ajuga turkestanica, a medicinal plant indigenous to Uzbekistan. Precursors of phytoecdysteroids (acetate, mevalonic acid cholesterol) or methyl jasmonate (an elicitor) were added to subculture media to increase phytoecdysteroid accumulation. In cell suspension cultures, 20-hydroxyecdysone (20E) content increased 3- or 2-fold with the addition of 125 or 250 μM methyl jasmonate, respectively, compared to unelicited cultures. Precursor addition, however, did not provoke phytoecdysteroid accumulation. In hairy root cultures, addition of sodium acetate, mevalonic acid, and methyl jasmonate, but not cholesterol, increased phytoecdysteroid content compared to unelicited cultures. Hairy root cultures treated with 150 mg l⁻¹ sodium acetate, or 15 or 150 mg l⁻¹ mevalonic acid, increased 20E content approximately 2-fold to 19.9, 20.4 or 21.7 μg mg⁻¹, respectively, compared to control (10.5 μg mg⁻¹). Older hairy root cultures, extracted after the seventh subculture cycle, also showed increases in 20E content (24.8 μg mg⁻¹), turkesterone (0.9 μg mg⁻¹) and cyasterone (8.1 μg mg⁻¹) compared to control cultures maintained for a shorter duration of four subculture cycles. Doses of 10 or 20 μg ml⁻¹ hairy root extract increased protein synthesis by 25.7% or 31.1%, respectively, in a C2C12 mouse skeletal cell line. These results suggest that sustainable production of metabolically active phytoecdysteroid can be achieved through hairy root culture systems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of Light on Mevalonic Acid Incorporation into the Phytosterols of Nicotiana tabacum L. Seedlings

Mevalonic acid-2-¹⁴C was readily incorporated into the free, esterified, and glycosidic sterol fractions of tobacco (Nicotiana tabacum L. var. Burley 21) seedlings. The time course of mevalonic acid-2-¹⁴C incorporation was different for the various individual sterols. Campesterol and sitosterol (group I) became radioactive as the free sterol and subsequently as the steryl ester. The reverse order was observed for cholesterol and stigmasterol (group II). Light stimulated the incorporation of mevalonic acid-2-¹⁴C into the group I free sterols and during the first 6 to 9 hours into the steryl esters of group II. The increase in specific radioactivity of the group II steryl esters was followed by a decline. Based on time course studies it is suggested that the group II steryl esters turn over rapidly and that light influences the rate of turnover.     

Casbene: An anti-fungal diterpene produced in cell-free extracts of Ricinus communis seedlings

The capacity of cell-free extracts of 2·5-day-old castor bean seedlings for synthesis of casbene from mevalonic acid were compared for seedings which had been germinated under sterile conditions and seedlings which were intentionally exposed to fungal cultures. Extracts from seedlings exposed to cultures of Rhizopus stolonifer, Aspergillus niger or Fusarium moniliforme produced much higher levels of casbene than extracts from sterile controls; the initial rates of casbene synthesis were 20–40 times higher in the extracts of seedlings that had been exposed to fungus. Although some variation in the capacity for synthesis of other diterpene hydrocarbons from mevalonic acid was seen in the two types of extracts, no consistent or striking stimulation in the synthesis of any of these was noted under these conditions of exposure of the seedlings to fungi. The potato-dectrose agar used as a fungal growth medium did not itself evoke the increase in casbene synthesis. Intact mycelia and cell-free extracts of mycelia of Rhizopus stolonifer gave no indication of diterpene biosynthesis from mevalonic acid. Purified casbene at concentrations of 10 μg ml⁻¹ or greater retarded the development of A. niger on potato-dextrose medium. Casbene was also found to inhibit the endogenous and gibberellic acid-stimulated growth of leaf sheaths of the dwarf-5 mutant of Zea mays and of the growth of the K-12 strain of Escherichia coli on glucose-minerals medium. It is suggested that casbene may serve the castor bean plant as a phytoalexin.     

The solubilization of carotenogenic enzymes of Phycomyces blakesleeanus

The effect of nine ionic and nine non-ionic detergents, over a 0.3–3.0% (w/v) concentration range, on the activity of the enzymes which convert [2-¹⁴C]mevalonic acid into phytoene (7,8,11,12,7′,8′,11′,12′-ψ,ψ-carotene) and β-carotene (β,β-carotene) has been investigated with cell extracts of the C115 carS42 mad-107(?) (β-carotene-accumulating) strain of Phycomyces blakesleeanus. The enzymes catalyzing the conversion of mevalonic acid into phytoene in the C115 and the C5 carB10(?) (phytoene-accumulating) strains of Phycomyces could be released from membranes with high molarity Tris-HCl buffer, but the other carotenogenic enzymes required solubilization with detergents. Enzymic activity was retained with only two ionic detergents (Zwittergents 3–8 and 3–10), whilst Tweens 40 and 60 were the least inhibitory of the non-ionic surfactants. Both Tween 60 and Zwittergent 3–08 solubilized almost 50% of the enzymic activities for the conversion of phytoene to β-carotene, but the former preparation was significantly more stable on storage at ?70°C.     

Metabolism of mevalonic acid to phosphorylated intermediates in a cell-free extract from Nepeta cataria leaves

A cell-free extract has been prepared from leaves of Nepeta cataria plants which converts mevalonic acid (MVA) to mevalonic acid phosphate (MVAP), mevalonic acid pyrophosphate (MVAPP) and isopentenylpyrophosphate (IPP). These enzymes are found in the 30 000 g supernatant. The activities are maximal at pH 7 and the formation of mevalonic acid pyrophosphate and isopentenyl-pyrophosphate reaches a maximal level after an incubation time of 180 min whereas the level of mevalonic acid phosphate begins to decrease after 90 min.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^?8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^?7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^?5, 5 × 10^?6, and 5 × 10^?7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^?8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1′-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.