Geranyl diphosphate synthase in leaves of Pelargonium roseum

Phyr~~misf~~l,Vol. 30, No. 6. pp. 1757-1761,1991 Pnnted in Great Bntain.

GERANYL

c

0031 9422,191 %3.OOe0.00 1991Pergamon Press plc

DIPHOSPHATE SYNTHASE IN LEAVES OF PELARGONIUM ROSEUM TAKAYUKI

and

SUGA*

TAKASHI

ENDO

Department of Chemistry, Faculty of Science, Hiroshima University, Higashisenda-machi, Naka-ku, Hiroshima 730, Japan

Key Wool Index- Prfargonium roseum; Geraniaceae; prenyltransferase.

geranium;

leaves; geranyl

diphosphate

synthase;

Abstract-A chain-length-sp~ific geranyl diphosphate synthase was detected in leaves of Pe~urgu~i~m roseurn. The enzyme systems, farnesyl diphosphate synthase [EC 251.11, geranylgeranyl diphosphate synthase and isopentenyl diphosphate isomerase [EC 5.3.2.21, were also detected. Furthermore, geranyl diphosphate synthase was separated from farnesyl diphosphate synthase by hydrophobic chromatography on Butyl-Toyopearl 650. This separation demonstrates that there are two specific synthases, geranyl diphosphate synthase and farnesyl diphosphate synthase, in this higher plant. The geranyl diphosphate synthase was locaiized in a subcellular membrane fraction rather than in the soluble fraction. The synthase was activated by Mn2 + rather than Mg”.

INTRODUCTION

RESULTS

In higher plants, geranyl diphosphate (GPP) is a significant precursor for the biosynthesis of many monoterpenoids [ 1,23 and the biosynthesis of GPP was investigated in several higher plants [3-61. However, an enzyme which specifically synthesized GPP from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) was not reported and the separation of GPP synthase from farnesyl diphosphate (FPP) synthase has not been achieved so far. Therefore, it is usually considered that GPP is formed by FPP synthase and that a specific GPP synthase does not exist [7--.9]. However, a GPP synthase has been found in microorganisms [ 10,i l] and these organisms also contained geranylgeranyl diphosphate (GGPP) synthase. Recently, we reported data on the enzyme system from the leaves of Fe~urffo~i~rnroseurn which specifically synthesize GPP rather than FPP and we demonstrated the existence of a chain-length-specific GPP synthase in the enzyme system [ 121. At about the same time, Heide [ 131 and Croteau ez ul. [14] reported the existence of the GPP synthase in ~~?~usperrn~rnery~~ror~~z~~ceil cultures and Sa/uia c$ficinalis, respectively. Recently, Heide et al. [IS] reported the further purification and characterization of this GPP synthase, but the separation of GPP synthase from FPP synthase was not described. In order to demonstrate the existence of a specific GPP synthase we have tried to separate GPP synthase and FPP synthase and have chosen Pelargonium roseurn Bourbon, in which the major components of the essential oils are geraniol (36%) and citroneliol (34%), as the experimental plant material.

*Author to whom correspondence should be addressed. PHY

30:6-B

The crude enzyme system was prepared by centtifugation of an homogenate at 700 y followed by recentrifugation of the supernatant at 10000 y. The enzyme activity was assayed with [4-14C]1PP and DMAPP or GPP in the presence of 2 mM MnCI, or 2 mM MgCI, and the mixture incubated at 30” for 24 hr. In order to identify the reaction products, the allylic isoprenyl diphosphates were subjected to hydrolysis with alkaline phosphatase. The free alcohols were then analysed by HPLC on both a normal phase column and a reversed phase column. The prenyltransferase activity was mainly found in the pellets obtained at 700 and 10000 rjr,whereas the 10000 g supernatant contained only slight enzyme activity. The major enzymatic products from DMAPP and [4-“C]IPP was identified as geraniol (Fig. 1A and B). On the other hand, from GPP and [4-14C]lPP, farnesol and geranylgeraniol were the major products (Fig. 1C and D). The synthesis of GPP was activated by Mn*+ (O.l-1OmM) rather than Mg2 + in the crude enzyme system. With the above range of concentrations of these divalent metal ions, the synthesis of FPP was lower than production of GPP. These results suggest that the synthase which produced GPP is different from the FPP synthase and that a specific GPP synthase exists in the leaves of P. roseum. The activity in the supernatant could be increased by homogenization of the leaves with quartz sand (solubili~tion cu 30%). Moreover, the activity of the prenyltransferase was mostly retained (more than 90%) in an acetone powder of a 30 000 g pellet. However, the enzyme could not be solubilized from this acetone powder without addition of detergent as discussed in the following section. At the beginning of this work, the pellet obtained at 30 OOOg was used as the crude enzyme fraction, but this pellet could not be efficiently and

1757

T. SUGA and T. ENDO

1758

9

(B) lo

(E)

WAFP+@%P M92’

I

J

IO

20

L 0

FIN. I. Normal by mcubation

phase HPLC of [4-‘4C]IPP

indicate the radloactivity

chromatograms and DMAPP

of HPLC

reproducibly

prepared.

Consequently

Analysis

of the products and authentic samples. The products were obtained

or GPP with the 700~ pellet in the presence of Mn”

or Mg’+.

A-D

fractions. E and E’ indicate the absorbance at 210 nm of authentic samples;

solvent; (2). Imalool, (3). geranylgeraniol:

of the fresh leaves was used for further tion studies.

20

IO Rt / min

Rt / min

(4), farnesol; (5). geraniol; (6). isopentenol; (7). dimethylallyl

powder enzyme purifica-

an acetone

(I),

alcohol.

were identified on this basis. With the crude and purified enzyme fractions, the synthesized allylic isoprenyl diphosphates were clearly established as GPP, FPP and GGPP.

of producrs

The enzymatic reaction products, which were formed from [4-‘4C]IPP with DMAPP or GPP, were hydrolysed with hydrochloric acid or alkaline phosphatase. After separatton of the hydrolysed products by HPLC together with an authentic sample of the alcohol. radioactivity was measured by liquid scintillation counting. The major products formed from DMAPP and [4‘JC]IPP were gcraniol and geranylgeraniol. On the other hand, the major product formed from GPP and [414C]IPP was farnesol. When the products were hydrolysed with hydrochloric acid. the primary alcohol and the tertiary alcohol were derived from the same primary allylic diphosphate. However, the unmetabolized [4‘JC]IPP was not hydrolysed by hydrochloric acid treatment. In the case of purification on reversed phase HPLC (see Experimental), radioactivity of the primary and the tertiary alcohol was measured together because the retention times of the two alcohols arc similar. To confirm the analysis of the products, the alcohols obtained by hydrolysis with alkaline phosphatase were separated on both normal phase and reversed phase HPLC columns. On a normal phase column, farnesol and geranylgeraniol could not be fully separated (see Experimental). The amounts of radioactivity which accompanied geraniol. farnesol and gcranylgeraniol on both the columns wcrc compared and then the product alcohols

Solubilization

of GPP synthasr

Attempts to solubilize the GPP synthase from the acetone powder were made with Triton X-100, Tween 80 or Tween 20. Triton X-100 was the most successful for solubilization and retention of activity of GPP synthase. When the solubilization was performed for 30 min at O‘, the solubilization was ca 40% with O.l% Triton X-100 and ca 70% with 5% Triton X-100. Higher concentrations of Triton X-100 did not improve the solubilizatton of the enzyme. When the soluhih7ation procedure was extended for 3 hr, the activity in the supernatant was somewhat decreased. The relatively high concentration of Triton X-100 present in the solubili7ed fraction caused foaming and posed a problem for the further purification of the enzyme, it was necessary to remove the Triton X100 with minimal loss of the enzyme activity. It has been reported that Bio-Beads SM-2 are useful for the removal ofTriton X-100 from protein solutions [lb]. However, in our case, after application of this method the enzyme activity was significantly decreased and the enzyme fraction diluted. Therefore. to remove the Triton X-100 and concentrate the enzyme, the solubilizcd enzyme solution was subjected to acetone precipitation. Using this method, the GPP synthase activity was not appreciably decreased. However, IPP isomerase activity was decreased by nearly 80%.

Geranyl diphosphate synthase of Pelor~oni~mroseum Puri~curion

1759

of GPP synthase

The acetone precipitated fraction was purified on an anion exchange column of DEAE-Toyopearl or on an hydrophobic column of Butyl-Toyopearl (Figs 2 and 3). Hydrophobic chromatography on Phenyl-Sepharose CL-4B was employed in the previous reports Cl 3-l S] for the purification of prenyltransferase. In the case of ButylToyopearl column chromatography, an affinity interaction between a butyl ligand and prenyltransferase was expected because this ligand is similar to the isoprene unit. On DEAE-Toyopearl, GPP synthase, FPP synthase and GGPP synthase could not be separated from each other. However, the FPP synthase peak showed some tailing of activity which was not seen in the GPP synthase activity peak and this suggested that the two enzyme activities reside with different proteins (Fig. 2A and B). In a preliminary experiment using Butyl-Toyopearl column chromatography, the GPP synthase was eluted at the end of a linear gradient of ammonium sulphate, but

this was not considered appropriate for the purification of the enzyme. Therefore, the gradient was changed to use an exponential curve starting from 12% saturated ammonium sulphate (see Fig. 3). With this system, three enzyme activity peaks (A-C) were observed in assays with

6

Ku IPP

;: :: :: : : ; : : :

0

IO

20

30

40

20

40

60

80 Fr

100

120

140

60

No

Fig. 3. Hydrophobic chromatography of the acetone precipitate on a Butyl-Toyo~arl 650 column. -0 and (a) axis, enzyme activity with DMAPP and [4-‘*C]IPP; -.-C- and (b) axis, enzyme activity with GPP and [Ci4C]IPP, - - -, absorbance at 280 nm; -, concentration of ammonium sulphate.

DMAPP and [4-r4CJIPP, but two enzyme activity peaks (A and B) were observed with GPP and [4-‘4C]IPP as the substrates (Fig. 3). The analysis data of the enzymatic products are summarized in Table 1. In peak A, radioactive dimethylallyl alcohol was mainly produced from both combinations of substrates. In peak B, farnesol was the major product from GPP and [4-‘4C]IPP, but from DMAPP and [4-‘4C]IPP, geraniol and geranylgeraniol were also produced. In peak C, geraniol and geranylgeraniol were synthesized from DMAPP and [4-‘4C]IPP, on the other hand, from GPP and [4-“CJIPP, allylic isoprenol products were negligible. These results show that IPP isomerase occurred in peak A, FPP synthase was in peak B and GPP synthase was located in peaks B and C. However, GGPP synthase activity overlapped the GPP synthase activity. GGPP synthase produced only GGPP without accumulation of GPP and FPP as an intermediate [17-191. The enzyme which synthesized GPP cannot be GGPP synthase, but must be an independent GPP synthase.

DMAPP + [4-‘4C]IPP

GPP c [4-T]

0

DiSCUSSION

50

60

70

80

SO

0

Fr No

Fig. 2. Anion exchange chromatography of the acetone precipitate on a DEAE-Toyopearl 650 column. A, enzyme activity was assayed with DMAPP and [4-“*C]IPP, B, enzyme activity was assayed with GPP and [4-“‘CJIPP. -O--, radioactivity of geraniol; ---(I)--, radioactivity of farnesol; -A-, radioactivity of geranylgeraniol; ---, absorbance at 280 nm. The arrow indicates the starting point of the linear gradient.

GPP synthase was partially purified from P. roseum and clearly separated from FPP synthase employing a single Butyl-Toyo~arl column (Fig. 3). The separation of these enzymes was also reported very recently by Croteau et al. [14]. These results demonstrate that a specific GPP synthase exists in higher plants. On Butyl-Toyo~arl column chromatography, GPP synthase and GGPP synthase were eluted together. A similar observation was obtained with a microorganism [l I]. In this latter case, the GPP synthase fraction even after pu~fication 490fold still included GGPP synthase activity. In the present work, GPP synthase was solubilized with Triton X-100. However, without detergent the solubilization of the enzyme activity from acetone powder was only 6%. In the case of flavedo of Citrus sinensis,

T. Sua~

Table

I. Incorporation obtamed

and T.

ENDO

of [4-‘4C]IPP into allylic isoprenols by from Butyl-Toyopearl column chromatography DOHt

GOH

FOH

GGOH

(dpm)

(dpm)

@pm)

(dpm)

A A B B C c

812 722 81 130 60 49

21 II 723 136 2659 15

39 0 42 1069 58 69

23 15 625 40 794 38

(DMAPP+[4-‘4C]IPP) (GPP+ [4-“CJIPP) (DMAPP + [4-‘4C]IPP) (GPP+ [4-“‘C]IPP) (DMAPP+ [4-‘Y]IPP) (GPP + L4-Y]IPP)

was extracted

from

an acetone

powder

[9]. Our results indicate that the enzyme of P. roseum may exist in a subcellular fraction rather than in the cytosol and it may be tightly bound to a membrane. It has been reported that FPP synthase is located in microsomes from pig liver [20] and that FPP synthase could be solubilized with Tritdn X-100. The P. roseum GPP synthasc required Mn2’ rather than Mg 2+ for the formation of GPP. This property is diKerent from the FPP synthase of pumpkin [7] which requires Mg2 T rather than Mn2’. However, GPP synthase from cell cultures of Lithospermum rrythrorhizon was activated by Mg2 ’ [l3]. Preparation of an acetone powder and an acetone precipitation of protein were adequate procedures for the partial purification of the enzyme system from P. roseum. Using these methods. the activity of the prenyltransferase was not greatly decreased. However, if the temperature of the acetone solution was allowed to rise, the enzyme activity was rapidly lost. EXPERIMENTAL

Marerids. Pelargonium roseurn was obtained from our botanical laboratory’s garden. [4-“‘C]IPP (sp. act. I.8 GBq mmol-‘) was obtained from New England Nuclear. DMAPP and GPP were prepared according to the report of ref.

[211. Preparorion

o/

sparhuse. The fresh leaves (70g) of in a mortar for 20 min with liq. N,. The powder was further homogenized in a Waring Blendor with 70 g of polyvinylpolypyrrolidone in 300ml of 0.05 M TES [N-tns(hydroxymethyI)-2-aminoethanesulphonic acid] buffer (pH 7.5). The homogenate was then filtered through four layers of gauze and the filtrate centrifuged at 700 (/ for 10 min. The resulting supernatant was centrifuged at 10000 y for 30 min. The obtained supernatant and pellets were assayed for prenyltransferase activity. For some studies, the filtrate was centrifuged directly al 30 000 y to provide the pellet which was used for the preparation of an acetone powder (see below). Preparurion of‘ ucetone powders. (i) The leaves (15 g). after being ground to a powder in liq. N,, were suspended in 100 ml cold Me,CO (- 70‘) and the suspension stirred for 30 min at -70’. After filtration with suction. the residue was dried in GPP

P. roseum were ground

fractions

Fraction* and substrates

*Fractions A. B and C corresponds lo activity tThe products of the enzymatic reaction were treatment and the free allylic isoprenols analysed DOH, Dimethylallyl alcohol: GOH, geramol; geraniol.

prenyltransferase

the

peaks A, B and C in Fig. 3. hydrolyscd by hydrochloric acid by HPLC (see Experimental). FOH, farnesol; GGOH. gcranyl-

~‘(u‘uo. The resulting

acetone

powder

was used as the crude at 30 000 y prepared from 70 g of leaves was suspended in a small amount of TES buffer (CU IO ml)at 0-, Me,CO(lOO ml)at -70 wasslowly poured into the suspension. The acetone suspensron was then gently stirred for 15 min at -70 After centrifugation al IO 000~ for 30 min. the ppt. was dried rn C(ICUO. Soluhilnarion of lhe enzyme system. The 30000~ acetone powder was suspended in 50 ml of TES buffer containing 5% Tnton X-100 and 5 mM l,4-dlthiothreitol. The suspension was gently stlrrcd at 0’ for 15 min and then centrifuged at IO0 000 y for 60 min. The supernatant was used for further purification. Purificafion of‘ GPP synrhuse. Cold Me,CO (IO vols, - 70’) was added to the solubilized fr. After gentle stirring for 15 min at - 70 ‘, the ppt. of proteins was sepd by centrifugation at IO 000 g for 30 min at - 30 ‘. The precipitate was immediately freed of aCetone in IXCUO. The ppt. was dissolved m ca IO ml TES buffer. The enzyme solution was applied 10 a 2 x 70 cm column of DEAE-Toyopearl equdibrated with 0.05 M TES buffer containing 50 mM NaCI. At the start, the column was washed wtth I column vol. of TES bufTer containing 50 mM NaCl and then the enzyme was eluted with a linear gradient of 3 column vols of 50-800 mM NaCl in 0.05 M TES buffer. Frs of IO ml were collected. The enzyme solution containing 12% (NH,),SO, was applied lo a 1.6 x 30 cm column of Butyl-Toyopearl equilibrated with 0.02 M TES buffer containing 12% (NH,),SO,. The column was washed with 4 column vols of the same buffer. Profeins were eluted with an exponential curve gradient of I5 column vols of I2 0% (NH&SO, in 0.02 M TES buffer. Frs of 4 ml were collected. Assap o/GPP synthase. To 1.0 ml of the enzyme solution was added 0.5 ml of 0.05 M TES bulTer (pH 7.5) containing 20 mM KF, I5 mM iodoacetamide, 1 mM 1,4-dlthiothreitol and I mM MnCI, (when the frs of CC were assayed, iodoacetamide was not added) and the mixt. was incubated at 37’ for 24 hr with the substrates (4 x IO.’ mol [4-“‘C]IPP plus I x 10 ’ mol DMAPP or GPP). After incubation, 200~1 of 2 M HCI was added and hydrolysis was performed for 30 min at 37’. After hydrolysis, the products derived from the acid labile allylic diphosphates were extracted with pentane after addition of 300~1 of 2 M NaOH to produce an alkaline solution. The radioactivity of extracts was measured by hquid scintdlation counting. In the case ofhydrolysis with alkaline phosphatase, the

enzyme system. (ii) The pellet obtained

Geranyl

pH of the reaction

mixt. was adjusted

diphosphate

synthase

lo 9-10 with aq. NaOH

and alkaline phosphatase (30 units) was added further incubated at 37” for 24 hr.

and the mixt.

Analysis of enzymatic reaction products. The extracts which were coned to 100 ~1 by evapn of solvent under normal pressure were chromatographed by HPLC on a Waters Radial Pak Silica 10 p column (5 x 100 mm) with a solvent system of hexane-nBuOH (125:2) at a flow rate of 0.5 ml min- ’ and a Waters Radial Pak Cl8 10 g column (5 x 100 mm) with a solvent system of MeOH-H,O (4: 1) at a flow rate of 0.8 ml min _ ‘. The elution of enzymatic products with authentic samples of geraniol, farnesol and geranylgeraniol was monitored at 210 nm and frs were collected. The radioactivity of each HPLC fr. was measured by liquid scintillation counting.

Acknowledgements-We thank the Central Research Laboratories of Kuraray Co. Ltd for a gift of samples of geraniol, farnesol and geranylgeraniol. The present work was supported in part by a Grant-in-aid for Scientific Research No. 61430010 from the Ministry of Education, Science and Culture.

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roseum

1761

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B. (1987) Biochem. Biophys.

Acto

920,140. 20. Ishii, K., Sagami,

H. and Ogura,

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1635. 21. Davisson, Methods.

V. J., Woodside, A. B. and Poulter 110, 130.

Enzymol.

C. D. (1985)