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Enzymatic Preparation of I4c-Labeled Phytoene

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Enzymatic Preparation of I4c-Labeled Phytoene Enzymatic Preparation of i4C-Labeled Phytoene, Squalene, and Geranylgeranyl Pyrophosphate from [2-nC]Mevalonic Acid Gerhard Sandmann *, Willy Hilgenberg **, and Peter Böger * * Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Postfach 5560 D-7750 Konstanz ** Botanisches Institut, Universität Frankfurt Z. Naturforsch. 35 c, 927-930 (1980); received August 25, 1980 14C-Labeled Terpenoids, cw-Phytoene, Kaurene, Squalene, Geranylgeranyl Pyrophosphate Methods are described for an enzymatic preparation of 1 4 C-labeled terpenoids. With a cell-free system of a white mutant of Phycomyces blakesleeanus (Mucoraceae) [1 4 C]squalene and [ 1 4 C- m]phytoene can be synthesized from [2- 1 4 C]mevalonate. The application of norflurazon, a phenyl- pyridazinone herbicide, helps to increase the yield of squalene. Furthermore, the liquid endosperm of Echinocystis lobata (Cucurbitaceae) was used for the formation of either [14C(-)]kaurene from [ 14C]mevalonic acid or [ 1 4 C-/ram']geranylgeranyl pyrophosphate in the presence of Amo 1618. The hydrocarbons formed were purified by alumina-column chromatography and preparative thm-layer chromatography (TLC). Geranylgeranyl pyrophosphate was separated by DE-column chromatography followed by TLC. Introduction Materials and Methods The use of radioactively labeled precursors is a 1. Cultivation of Phycomyces blakesleeanus prerequisite for the study of terpenoid synthesis and Echinocystis lobata in vivo and in vitro. Geranylgeranyl pyrophosphate is The enzyme system converting [ 14C]mevalonic the common substrate for all C 20 and C 40 terpenoids. acid to squalene and phytoene was obtained from Its radioactivity can be channelled directly into a phytoene-accumulating mutant of Phycomyces carotenes, phytol, gibberellines, and tocopherols in blakesleeanus [strain C5-car-B 10(—)]. G row th was different microorganisms and plants [1,2]. Squalene, carried out as described [ 6 ] in a liquid aspar­ kaurene, and phytoene are the direct precursors for agine glucose medium in the light for 3 days on a all sterols, gibberellines, and carotenes. Their avail­ shaker. Immature seeds of E. lobata were the source ability as radioactively labeled substrates is very of the enzyme extract for geranylgeranyl pyrophos­ helpful in biosynthesis studies of the terpenoid phate and kaurene synthesis. E. lobata (Greene) was groups mentioned [3]. cultivated in the Botanical Garden of the Universität Furthermore, there is a need for [ 14C]geranyl- Frankfurt. Seedlings were grown from fruits col­ geranyl pyrophosphate as a reference compound lected at the natural habitat near Ladenburg. Prior when the mode of action of growth retardants and to sowing, the seeds were stored at 4°C from au­ herbicides on kaurene synthetase [4] and phytoene tumn to spring. synthetase [5], respectively, is studied. None of the substances mentioned is available commercially as a 2. Preparation o f14C-cis-phytoene and 14C-squalene labeled compound. using Phycomyces The present paper describes methods of how labeled squalene, (-)-kaurene, cw-phytoene, and The enzyme extract for the biosynthesis of [ 14C-m ]- a//-rra«£-geranylgeranyl pyrophosphate can be pre­ phytoene and [ 14 C]squalene was prepared from 2 g pared from [2- 14 C]mevalonic acid with cell-free sys­ of the freeze-dried mycelium ground in a Dangoumau tems from a fungus and the seeds of a higher plant, mill (type 1241, Sovirei, Wiesbaden) with a nylon both which can be easily cultivated. vessel and korund balls, and 7 ml of 30% glycerol in 0.2 M Tris-HCl buffer [N-tris(hydroxymethyl)amino- methane], pH 8.0, containing 2 m M mercaptoethanol. Reprint requests to Prof. Dr. P. Böger. After centrifugation at lOOOOx# for 30 min, the 0341-0382/80/1100-0927 $01.00/0 supernatant was used immediately. The reaction was 928 G. Sandmann et al. ■ Enzymatic Preparation of 14C-Labeled Terpenoids carried out anaerobically in Thunberg tubes over a development, the liquid endosperm is not sur­ period of 5 h at 24 °C. At start, evacuation of the rounded by the cotyledons. The seeds were cut open tubes and flushing them five times at least with and the endosperm was squeezed out and centri­ nitrogen is necessary for a satisfactory yield of fuged for 30 min at 20000 xg. The supernatant was squalene. The reaction medium, modified after [5,7], used as the (enriched) enzyme extract. The reaction consisted of 0.1 ml o f 0.3 mM DL-[2- 14C]mevalonic mixture for the assay according to [9] contained: acid, sodium salt (CEA France, specific activity 0.1 ml of 20 m M ATP; 0.1ml of 20 m M M gS 04; 32 mCi/mmol); 0.1 ml of 40 mM MgCl 2 and 40 mM 0.2 ml o f 0.1 M Tris-H Cl buffer, pH 7.0; 0.1 ml o f [2- MnCl2; 0.2 ml of cofactor solution (= glutathione, 14C]mevalonic acid; and 1 ml of enzyme extract con­ lOOmM, ATP; 50 mM, N A D P; 5 mM; N A D , 5 mM; taining 3.5 mg of protein. After a 1 h reaction time at FAD, 5 mM, dissolved in the buffered extraction 30 °C saponification, extraction of [ 14C]kaurene, medium), and 0 . 6 ml of the enzyme-containing and separation on alumina columns and by TLC supernatant. The protein content of the 1 ml reaction were carried out as described for phytoene and mixture was 12 to 13 mg. To some incubation squalene. samples 10 hm of norflurazon (4-chlor-5-[methyl- amino]-2-[3-trifluoromethylphenyl]-pyridazin-3[2//]- 4. Preparation of f 4C]trans-geranylgeranyl one) was added. For the extraction of radioactive pyrophosphate compounds 1 ml of aq. KOH solution (60%, w/v) and 8 ml of ethanol, the latter containing 50 |ig of The same reaction mixture as for the kaurene unlabeled carrier phytoene and squalene, were preparation was used for geranylgeranyl pyrophos­ added to the incubate, then the mixture was saponi­ phate synthesis, but additionally 5 m M A m o 1618 fied over night at room temperature or for 1 h at [2'-isopropyl-4/-[trimethylammonium chloride]-5'- 70 °C. Cw-phytoene was isolated from the same methylphenyl-piperidine- 1 -carboxylate] was present. strain of Phycomyces according to [ 8 ]. The solution After a 3 h reaction time, the whole mixture was was extracted 3 times with ethyl ether. The organic boiled for 2 min and centrifuged at 1 0 0 0 0 x g for phase was washed with water, dried with Na 2S 0 4, 10 min. The pellet was extracted with some ml of and evaporated in a stream of nitrogen. The residue water and the homogenate centrifuged again. The was dissolved in light petrol (b. p. 40-60 °C) and combined supematants were placed on top of a placed on a column of alumina (1.5x50cm; Whatman DE-52 cellulose column (1.8x12 cm) Woelm, Eschwege, neutral, grade III). Phytoene and equilibrated with water [10], Elution was carried out squalene were eluted with petrol (40-60 °C) with a linear gradient of 0 to 0.2 M (NH 4) 2C 0 3. The and purified further by thin-layer chromatography absorbance at 265 nm was recorded and 10 ml frac­ on silica gel G plates (Merck, Darmstadt) with tions were collected. The ATP appeared in samples petrol (b. p. 60-80 °C) [7]. The compounds were No. 40 to 45. The following 5 samples were pooled detected by spraying with a solution of rhod- and freeze-dried after adding some Dowex 50 (H+) am ine 6 G in acetone (1% w/v) and examining the to remove the NH4+ from the solution. [ 14C]geranyl- plates under UV light. Squalene separated at Rf geranyl pyrophosphate was further purified by TLC values between 0.45 and 0.6, phytoene between R f of on silica gel plates in a solvent system of isopro- 0.35 and 0.45. Purity of both labeled compounds was panol/ammonia (25%)/water (60:30:10, v/v) [11]. checked by running squalene on silica gel G plates The radioactivity of geranylgeranyl pyrophosphate with cyclohexane/chloroform (50:50, v/v) and was located in a zone between R f 0.7 and 0.8. For a phytoene on silica gel G plates in a system of 0.5% definite identification of geranylgeranyl pyrophos­ (v/v) benzene in petrol (b. p. 60-80 °C). phate, aliquots were hydrolyzed with alkaline phos­ phatase [10] and the resulting [ 14C]geranylgeraniol co-chromatographed with an authentic standard of 3. Preparation of f 4C-(-)J kaurene pyrophosphate geranylgeraniol on silica gel plates using benzene/ using Echinocystis lobata ethylacetate (80:20, v/v) as a solvent [11], The spots For the kaurene and geranylgeranyl pyrophos­ of kaurene, geranylgeraniol, and geranylgeranyl phate preparation soft immature seeds were em­ pyrophosphate were detected by staining with iodine ployed which were not yet colored. At this stage of vapor. The radioactivity on the plates was traced G. Sandmann et al. • Enzymatic Preparation of 14C-Labeled Terpenoids 929 with a thin-layer radioscanner (Berthold, Erlangen, Table II. Formation of 1 4 C-labeled (—)-kaurene and trans- geranylgeranyl pyrophosphate by an enzyme extract from LB 2722). Liquid samples were assayed in a liquid Echinocystis lobata. scintillation spectrometer (Packard Tricarb 3320). Protein was determined with the Folin reagent [12]. Products Radioactivity [dpm] 5. Chemicals No addition Amo 1618 Geranylgeraniol was a gift of Dr. W. Rüdiger, (—)-kaurene 385000 1 2 0 0 0 München, (-)-kaurene was a gift of ICI, Eng­ /ra/w-geranylgeranyl pyrophosphate 2 1 0 0 0 230000 land. Alkaline phosphatase, ATP, NADP, NAD, FAD, and glutathione were purchased from Boehrin­ ger, Mannheim. Other chemicals were from Merck, Darmstadt, unless stated otherwise. Squalene was purchased from Sigma, München, norflurazon was from Sandoz AG, Basle. Results and Discussion In this communication we describe the prepara­ tion of several radioactively labeled terpenoids.
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