In spinach chloroplasts, 1,4-dihydroxy-2-naphthoate is prenylated by phytyldiphosphate and subsequently methylated by S-adenosylmethionine to form phylloquinol. The site of the prenylation reaction is the chloroplast envelope membrane

Baldwin

Block

Bryant

Dialameh

Douce

Haas

Hutson

Jensen

Lichtenthaler

Martius

Mayer

Rüdiger

Shineberg

Soil

Soll

Suttie

Thomas

Young

Zenk

English

Gernot SCHULTZ

Jürgen SOLL

Bernd H. ELLERBROCK

Crossref

European Journal of Biochemistry

Site of Prenylation Reaction in Synthesis of Phylloquinone (Vitamin K<sub>1</sub>) by Spinach Chloroplasts

Schultz, Gernot

Soll, Jürgen

Ellerbrock, Bernd H.

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Eur. J. Biochem. 117,329-332 (1981) Q FEBS 1981 Site of Prenylation Reaction in Synthesis of Phylloquinone (Vitamin K,) by Spinach Chloroplasts Gernot SCHULTZ. Bernd H. ELLERBROCK, and Jurgen SOLL Institut fur Tierernahrung, Arbeitsgruppe fur Phytochemie und Futtermittelkunde, and Botanisches Institut, Tierarztliche Hochschule, Hannover (Received December 29, 1980/March 24, 1981) In spinach chloroplasts, 1,4-dihydroxy-2-naphthoate is prenylated by phytyldiphosphate and subsequently methylated by S-adenosylmethionine to form phylloquinol. The site of the prenylation reaction is the chloro- plast envelope membrane. 1,4-Dihydroxy-2-naphthoate [1, 21 as the crucial inter- mediate in the biosynthesis of menaquinones in bacteria is formed from shikimate [3] via chorismate [2] and o-succinyl- benzoate [I]. Prenylation [4] and methylation [I] result in formation of menaquinones of different length of side chain. The principal steps of the synthesis of the naphthoquinone moiety are similar for both plants and bacteria [5 - 71. In the present paper we wish to report on the prcnylation of 1,4-dihy- droxy-2-naphthoate by phytyldiphosphate and subsequent methylation by S-adenosylmethionine to form phylloquinol in spinach chloroplasts. The site of the prenylation reaction is the chloroplast envelope membrane. MATERIALS AND METHODS Radiochenzira1,s and Chemical Synthesis S-[methyl-'4C]Adenosylmethionine (56 Ci/mol) was pur- chased from Amersham Buchler (Braunschweig, FRG), [U-14C]phytol (80 Ci/mol) from NEN (Dreieich, FRG). [U-'4C]Phytyl diphosphate was prepared according to [8] modified as follows : 0.75 mg (z 200 pCi) of the above labeled phytol dissolved in 25 p1 acetonitrile was added dropwise within 2 h by a micropipette to a 250-pl reaction vessel con- taining 100 p1 di(triethy1ammonium) phosphate (3.3 % in dry acetonitrile) and 10 pl trichloroacetonitrile. After leaving overnight at 0°C in the dark, phytyl phosphates were precipitated by adding 100 pl acetone plus 5 pl concentrated NH3. After 2 h at - 20 "C the precipitate was washed twice with 100 $0.28 M NHJ in methanol to remove phytyl mono- phosphate. The yield of the diphosphate was 30 pCi. 1,4-Di- hydroxy-Znaphthoic acid was prepared from 1 -hydroxy- 2-naphthoic acid according to [2], 1,CNaphthoquinones and 2-methyl-3,4-naphthoquinones with phytyl, geranylgeranyl, farnesyl and geranyl side chain, respectively, (2-prenyl-l,4- naphthoquinones and 2-methyl-3-prenyl- 1 ,Cnaphthoquino- nes) were synthesized as described under 'synthesis of mena- quinones, general procedure' in [9] modified as follows. Tetrahydrofuran was used as solvent with BFJ etherate. The etheral solution of the prenylnaphthoquinol was shaken with Dedicated to Prof. Dr Helmut Holzer, Freiburg/Br., on the occasion - _ _  of his 60th birthday. aqueous solution of sodium dithionite and was alkalinized by NAOH. After oxidizing the quinol by AgzO in the dark in dry diethylether, the product was chromatographed on a column of silicagel (1.5 x 40 cm) with light petroleum/diethyl- ether (20- 10: 1) as solvent. If necessary, the product was rechromatographed with the same system on thin-layer. For chloroplast experiments, prenylated and non-prenylated naph- thoquinols were formed by reduction from the corresponding quinones just prior to use: some crystals of NaBH4 were added to a mixture containing 50 p1 of the 1 mM quinone, 2.5 ml methanol, 2.5 ml diethyl ether and 0.5 ml water. After 1 min the quinol was extracted with 10 ml diethyl ether, washed three times with 5 ml water each and quanti- fied photometrically (approximated E243nm 27 500 mol-' 1 ern-', see [9,103). The required amount was evaporated under nitrogen in the incubation vessel of the chloroplast experi- ments. Isolation of Broken Chloroplasts Broken chloroplasts were prepared from intact chloro- plasts isolated according to [ l l ,  121; however, the concen- tration of MgClz in all the media [ I l l  was 2 mM, MnCb 1 mM and EDTA 1 mM. The chloroplast pellet which re- sulted from centrifugation of suspension in the modified medium A [l I ]  was suspended in the modified medium C 11 11 (about 2 mg chlorophyll/ml medium C). The chloroplasts were further purified on a linear Percoll gradient (Pharmacia, Uppsala, Sweden) (3500 x g  at 0°C for 45 min) [12]. The lower of the two bands contained the purified intact chloro- plasts. To remove adhering Percoll, the intact chloroplasts were washed twice with modified medium C [Il l .  To obtain 'broken chloroplasts', the above intact ones were osmotically shocked (1 ml water per > 1 mg chlorophyll for 20 min). Isolation of Chloroplast Subfractions Chloroplast subfractions were isolated from the pellet of the above intact chloroplasts after purification by a Percoll gradient. After an osmotic shock, thylakoid and envelope membranes as well as stroma phase were separated on a sucrose gradient according to [I41 (see also [13J). For criteria of purity of subfraction see [14]. 3 30 Reaction Mixtures in Chloroplast and Subfraction Experiments Reaction mixtures in experiments in Tables 1-3 and Fig. 1 - 2  contained the substrates and broken chloroplasts (or chloroplast subfractions) suspended in 1 ml of medium C according to [ l l ]  which was modified as mentioned above (2mM MgClz; 1 mM EDTA). The concentrations of the substrates added were, if not otherwise defined : 1 ,Cdihydroxy- 2-naphthoate 5 pM (1 pI of the 5 mM acid in ethanol was added to the reaction mixture) ; 2-methyl-I ,4-naphthoquinol 5 pM; ATP 0.2 mM; [U-'4C]phytol 12 pM; S-[m~thy l - '~C] -  adenosylmethionine 16 pM (for further details see tables and figures). Reaction mixtures were incubated at 20 "C for 80 min. The light intensity of the illumination was 0.1 J x cmp2 x s- ' .  Aliquots (200-300 pl) were taken at different times. The reaction was stopped and the lipids extracted as described in [13]. Protein contents were determined by the method of Lowry and chlorophyll by the method of Arnon. Purfication and Identification of Labeled Products Unlabeled carrier substances (about 25 pg per aliquot) identical to the expected labeled products were added to the extraction solution. All the naphthoquinols were oxidized for better purification before chromatography. The resulting 2-prenyl-l,4-naphthoquinones and 2-methyl- 3 -prenyl-1,4- naphthoquinones were chromatographed on silica gel layers with light petroleum (b.p. 60-80 "C)/diethyl ether (10: 1) as sol- vent and rechromatographed on cellulose layers (Schleicher & Schull, Einbeck-Dassel, FRG), impregnated with 7 paraffin oil in petrol (b.p. 100 - 140 C) with aceton/methanol/HzO as solvent. The solvent system in the case of phytyl derivatives were 13 : 7 : 0 5, with geranylgeranyl derivatives 13 : 7 : 1, with farnesyl derivatives 13 : 7 : 2 and with geranyl derivatives 13 : 7 : 3. Non-prenylated naphthoquinones were chromato- graphed on silica gel layer with light petroleum/diethyl ether (5 :I) and rechromatographed on silica gel layers with CHC13/ cC14 (1 : I )  as solvent. ,411 the SUbStdnCeS were cochromato- graphed with authentic samples and detected by ultraviolet quench at 254nm. Labeled products were counted as pre- viously described [13]. Table 1 .  Incorporation of I4C from [ U-'4CJphytol plus ATP into 1,4-di- hydrox~.-2-nuphthoair by .xpinach chloroplasts 1.2 mg chlorophyll per experiment (1 ml); for further details see Materials and Methods Incub,ition with 2-[phyr~~l-'~C]Phytyl 1,4-naphthoquinol formed in the light dark pmol x mg chlorophyll ' x h  ' -~ ~~ - ~P ~~ ._ 1,4-Dihydroxy-2-naphthoate 1,4-Dihydroxy-2-naphthoate [U-'4C]Phytol + ATP 1 0  0 5  + [U-14C]phytol + ATP 28 0 3 0  + [U-'4C]phytol 43 5 1 5  Formation of 2-Phytyl-1,Cnaphthoquinol ,from I ,4-Dihydroxy-2-naphfhoate in Spinach Chloroplasts Adding phytol in the light, 2-phytyl-1 ,4-naphthoquinol was formed from 1,4-dihydroxy-2-naphthoate by purified chloroplasts, which were slightly osmotically shocked in concentrated suspension ( > 1 mg chlorophyll/ml). Maximal rate obtained was 63 pmol x mg chlorophyll-' x hp'. The rate was not enhanced by adding ATP to the suspension. An active kinase which pyrophosphorylates phytol is located in the stroma of the chloroplast [13]. Thus, it may beconcluded that only phytol but not ATP was transferred across the slightly shocked envelope membrane: addition of ATP in the dark had minimal effects (Table 1). ATP required for pyrophos- phorylation of phytol was probably formed by photophos- phorylation of chloroplast thylakoids in the light. The mechanism of prenylation of the naphthalenic moiety resembles that of bacteria, but differs from animals, which are capable of forming menaquinones from 2-methyl-l,4- naphthoquinone (or the quinol) plus prenyl diphosphate [26, 171. In chloroplasts 2-methyl-1 ,Cnaphthoquinol in not preny- lated by phytol plus ATP in the light nor is 1,4-dihydroxy- 2-naphthoate methylated by S-adenosylmethionine (Table 2) .  Phylloquinol Formation,from 2-Phytyl-l,4-naphthoquinol RESULTS in Spinach Chloroplasts quinol formation were identified : In spinach chloroplasts the last two steps of phyllo- When S-adenosylmethionine was additionally applied in experiments with 1,4-dihydroxy-2-naplithoate plus phytol, WcooH +phytyl-pp, - +AdoMet \ /  PPPi, -co* \ -AdoHcy ' PhYtYl ' phytyl OH OH OH 1.4-Dihydroxy- 2-Phytyl- Phylloquinol -2-naphthoic acid -1.4-naphthoquinol Scheme. The lust two .steps of the formation o f  phylloquinol. Phytyl-PP, phytyl diphosphate; AdoMet, S-adenosylmethionine; Ado-Hcy, S-adenosyl- homocysteine The mechanism is analogous to bacteria; however, only the phytyl moiety (from phytyl diphosphate) is used in the prenylation reaction. According to the tocopherol synthesis [I 51 only the quinol and not the quinone was prenylated. phylloquinol was formed along with 2-phytyl-I ,4-naphtho- quinol (Table 3). Thus, the intermediately formed 2-phytyl- 1,4-naphthoquinol reacted with S-adenosylmethionine to form phylloquinol. Furthermore, when 2-phytyl-l,6naphtho- 331 h L7 4 N 2 -  Table 2. Incorporation of I4C jrom [ U-'4C]ph~~toi and S-[~nethyl-~~C]-  adenosylmethionine, respectively, into 1,4-dihydroxy-2-naphthoate, and the same for [U-'4C]phytol into 2-methyl-1,l-naphthoquinol by spinach chloro- plasts 1.17 mg chlorophyll per experiment (1 ml); for further details see Materials and Methods *-• o k =  - - B Incubation conditions Product formed ~- ~ ~~ tY Pe amount pmol x mg chlorophyll-' x h- '  1,4-Dihydroxy-2-naphthoate 2-[ph~~tyl-'~C]phytyl- + [U-'4C]phytol 1,4-naphthoquinoI + ATP; light 63 1,4-Dihydroxy-2-naphthoate 2-[methyl-'4C]methyl- + S-[mefhyl-'4C]adenosy1- 1 ,4-naphthoquinol methionine; dark 3.5 2-Methyl-1,4-naphthoquinol [phytyl-14C]phyllo- + [U-14C]phyt~l  quinol + ATP; light 2.5 Table 3. Incorporation of I4C,from [U-'4C]phytol and S-udenosylmezhionine into I ,4-dihyduoxy-2-naphthoate by spinach chloroplasts 1.3 mgchlorophyll per experiment (1 ml); for further details see Materials and Methods c "1 \t J Incubation with S-Adenosyl- Formation of methionine ~ ~ - ~ ~ 2-[phytyl- [phytyl- '4CI- I%]- phytyl- phyllo- 1,4-ndph- quinol thoquinol PM pmol x mg chloro- phyll-' x h- '  _ _  _ _  1,4-Dihydroxy-2-naph- thoate + [U-'"CJ- phytol + ATP - 22 5 0 15 10 21 0 0 45 100 24 9 1 0 5  [U-'4C]Phytol + ATP - 0 3  0 10 quinol (5 pM) was used as substrate, it was methylated by S-adenosylmethionine to yield phylloquinol. The maximal rate obtained was 6 pmol x mg chlorophyll-' x h-'. Increasing the applied concentration of 2-phytyl-I ,4-naphthoquinol from 1-5 mM, enhanced the phylloquinol formation in chloro- plasts only slightly. Consequently, the reaction might be controlled by diffusion of substrate across the envelope membrane. In testing the substrate specificity, the 2-phytyl- I ,Cnaphthoquinol was preferred to the geranylgeranyl de- rivative (ratio of phylloquinol : menaquinol-formation was (1 : 0.3). Triton X-100 (0.2 x )  (see [4]) exhibited no stimulatory effect on the methylation reaction. Elimination of an endog- enous byproduct, which is methylated at high rates at the thylakoids was a technical problem in determining the methylation rate. For this reason, experiments using farnesyl and geranyl derivatives were not conclusive. Synthesis of 2-Phytyl-1,l-naphthoguinol by Chloroplusts Subfractions In applying phytol plus ATP in the dark, only envelope membranes are active in synthesizing 2-phytyl-l,4-naphtho- quinol from 1,4-dihydroxy-2-naphthoate. The required phytyl diphosphate is formed by the kinase in the stromal phase [I 31, which was added to the envelope membranes in the test. Neither thylakoid membranes nor stromal protein are capable of forming the quinol (Fig. 1). In the experiment in Fig. 2, phytyl diphosphate instead of phytol plus ATP was applied. In this system the optimal substrate concentrations for prenyltransferase reaction in en- velope membranes are : 1 pM 1,4-dihydroxy-2-naphthoate and about 3 pM phytyl diphosphate. Equal rates are obtained with 20 pM phytyl diphosphate [56 pmol x (mg envelope pro- tein)-' x h-']. 1mM MgC12 instead of 2 mM MgClz reduces the rates by about the half. (Mg2+ concentration optimum of the reaction is at  2 mM ; data not shown.) Thylakoid mem- branes are also inactive in the system using phytyl diphosphate. DISCUSSION Phylloquinone and generalized prenylquinone synthesis in chloroplasts (e.g. the synthesis of tocopherols, plastoquinone and phylloquinone) [I 31 exhibits a sequence of coordinated reactions starting with the synthesis of the quinone moiety [I81 and of isopentenyl diphosphate in the stromal phase to the synthesis of the prenylated quinones at the membranes, preferentially the envelope membrane [I 3,191. The group of Douce demonstrated [20] that the prenylpyrophosphates are formed from isopentenyl diphosphate by a soluble transferase of the stroma which is effectively activated by chloroplast membranes, both envelope and thylakoid membranes. Fur- 332 I 2 40 ._ 3 CT 0 LL a m cc 's. 20 c - A A c L 4 c1 '0 20 40 60 80 Time ( m i n )  Fig. 2. Formation of 2-(phytyl-'1C]ph)~ryl-l~4-naphtho~uinol from 1,4-di- hydroxy-2-naphihoate plus [U-'4C]phytyl diphosphate (3  p M ) .  Filled symbols: envelope membranes (1.25 mg protein/ml) (A) 1 pM 1,4-di- hydroxy-2-naphthoate, 2 mM MgClz ; (W) 10 pM 1,4-dihydroxy-2-naph- thoate, 2 mM MgClz; (*) 10 pM 1,4-dihydroxy-2-naphthoate, 1 mM MgC12; (@) 0.1 mM 1,4-dihydroxy-2-naphthoate, 2 mM MgClz. Open symbols: thylakoid membranes (9.15 mg protein/ml; 1.05 mg chloro- phyll; (A) 1 pM 1,4-dihydroxy-2-naphthoate, 2 mM MgC12; (0) 10 pM 1,4-dihydroxy-2-naphthoate, 2 mM MgClz; (a) 10 pM 1,4-dihydroxy- 2-naphthoate, 1 mM MgC12; (0) 0.1 mM 1,4-dihydroxy-2-naphthoate, 2 mM MgClz. For further details see Materials and Methods thermore, most of the prenyl diphosphate is dispersed in membranes [20]. Thus, during biosynthesis the isoprenidic tails of the diphosphates may be positioned in membranes. In chloroplasts, however, phytyl diphosphate is used in the synthesis of the prenylquinones a-tocopherol [13] and phyllo- quinone, whereas geranylgeranyl diphosphate is preferred in the synthesis of chlorophylls [21]. The hydrogenation of geranylgeranyl diphosphate to yield phytyl diphosphate [22] may occur in membranes. As with plastoquinone and toco- pherol synthesis [I 31, the present work reveals that also in phylloquinone synthesis the prenylation step occurs in the envelope membrane : 2-phytyl-1 ,4-naphthoquinol is formed from 1,4-dihydroxy-2-naphthoate and phytyl diphosphate at an optimal concentration range of 1 pM each. Phylloquinones are located in thylakoid and envelope membranes both [23], G. Schultz and J. Soll, Institut fur Tierernahrung, Tierarztliche Hochschule, Bischofsholer Damm 15, 3000 Hannover 1, Federal Republic of Germany B. H. Ellerbrock, Botanisches Institut, Tierarztliche Hochschnle, Bunteweg 17d, 3000 Hannover-Kirchrode, Federal Republic of Germany but at present its function in plants is unknown and there is no indication if it acts in y-carboxyglutamate synthesis, as widely demonstrated in animals by Suttie et al. [24] and recently described in bacteria [25]. Financial support from the Deutsche Forschungsgemeinschaft is gratefully acknowledged. REFERENCES 1. Bryant, R. W., Jr & Bentley, R. (1976) Biochemistry, 15,4792-4796. 2. Young, I. G. (1975) Biochemistry, 14, 399-406. 3. Baldwin, R. M., Snyder, C. D. & Rapoport, H. (1974) Biochem- 4. Shineberg, B. & Young, I. G. (1976) Biochemistry, 15, 2754-2758. 5. Zenk, M. H. & Leistner, E. (1968) Lloydia (Cinci.) 31, 275. 6. Thomas, G. & Threlfall, D. R. (1974) Phytochemistry, 13, 807-813. 7. Hutson, K. G. & Threlfall, D. R. (1980) Phytochemistry, 19, 535- 8. Joo, C. N., Park, C. E., Kramer, J. K. G. & Kates, M. (1973) Can. 9. Mayer, H. & Isler, 0. (1971) Methods Enzymol. 18c, 491 -547. istry, 13, 1523 - 1530. 531. J. Biochem. 51, 1527-1536. 10. Lichtenthaler, K. H., Karunen, P. & Grumbach, K. H. (1977) Phy- 11. Jensen, R. G. & Bassham, J. A. (1966) Proc,. Nut1 Acad. Sci. USA,  12. Haas, R., Siebertz, H. P., Wrage, K. & Heinz, E. (1980) Planta 13. Soll, J., Kemmerling, M. & Schultz, G. 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in Biogenesis and Function of Planr Lipids

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Site of Prenylation Reaction in Synthesis of Phylloquinone (Vitamin K1) by Spinach Chloroplasts

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Site of Prenylation Reaction in Synthesis of Phylloquinone (Vitamin K1) by Spinach Chloroplasts

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