The Journal of Biochemistry, Vol. 64, No. 2, 1968

The Purification of Prenyltransferase and Isopentenyl Pyrophosphate Isomerase of Pumpkin Fruit and Their Some Properties

By KYOZO OGURA, TOKUZO NISHINO and SHUICHI SETO

(From the Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai)

(Received for publication, March 2, 1968)

Prenyltransferase ( synthetase) [EC 2.5. 1. l]

and isopentenylpyrophosphate isomerase [EC 5.3.3.2] were obtained and

partially purified from pumpkin fruit. The prenyltransferase preparation catalyzed the condensation of isopentenyl pyrophosphate with dime thylallyl pyrophosphate as well as with to yield

trans-traps farnesyl pyrophosphate as a final product, and was free of

isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthetase activities. Prenyltransferase of pumpkin has properties similar to those of pig liver, showing requirement of Mg++, Km value of 1.3•~

10-6 M for geranyl pyrophosphate, and pH optimum at 7.5.

Enzymatic formation of farnesyl pyro phatase [EC 3. 1.3. 11 of calf intestine was purchased from Boehringer and Soehne, Germany. Silica gel G phosphate which is an important precursor in the isoprenoid biosynthesis has been studied in and Kieselgur G were products of E. Merck, Germany. detail by using enzyme systems obtained from Geraniol, nerol and farnesol were generous gifts by Takasago Perfumary Co. Geranylgeraniol and geranyl yeast (1) and mammalian liver (2-6). It is linalool were kindly provided by Dr. Isler of Hoffmann known that farnesyl pyrophosphate synthetase - La Roche and Company, Switzerland. Sephadex G of pig liver catalyzes not only the reaction of 200 was a product of Pharmacia, Sweden. Hydro geranyl pyrophosphate with isopentenyl pyro xylapatite was prepared according to the method of phosphate but also the reaction of dimethylallyl TISELius et al. (11). Ion exchange resin was purchased pyrophosphate with isopentenyl pyrophosphate from Rhom and Hass, U.S.A. 1-14C-Isopentenyl pyro (3, 4). Isopentenyl pyrophosphate isomerase phosphate (1.2 ƒÊCi per ƒÊmole) was chemically synthe has also been isolated from the same organisms sized from methallyl chloride and 14C-barium carbonate (7-9). As to plant enzyme, however, only (Daiichi Pure Chemicals) essentially by the method of a single report of crude enzyme of carrot YUAN and BLOCH (12) except that the method of KANDUTSCH et al. (13) was employed for phosphoryl catalyzing the synthesis of geranylgeranyl ation of isopentenol. Purification of 14C-isopentenyl

pyrophosphate is found in the literature, in pyrophosphate was performed by gradient elution on which only the reaction of farnesyl pyrophos a column of Amberlite CG-400 formate in a similar phate with isopentenyl pyrophosphate is des way described by BLOCH et al. (14). The product cribed (10). was identified by paper chromatography on Toyo filter

The present paper reports the partial paper No. 51A in the system of n-propanol : ammonia : purification of prenyltransferase catalyzing water (6: 3: 1) (3, 10). Dimethylallyl, geranyl, and the formation of farnesyl pyrophosphate and farnesyl pyrophosphates were prepared by the pyro that of isopentenyl pyrophosphate isomerase phosphorylation of corresponding alcohols. Dimethyl from pumpkin fruit. Some of their enzymatic allyl alcohol was synthesized by the reduction of 3, 3- dimethylacrylic acid, which was synthesized from properties are described. mesity oxide, with lithium aluminium hydride (15). Assay for Isopentenyl Pyrophosphate Isomerase Activity MATERIALS AND METHODS -The reaction tubes contained, in a final volume of

Pumpkins were obtained locally. Alkaline phos 1.0ml, 25ƒÊmoles of phosphate buffer, pH 7.0, 5ƒÊmoles 197 198 K. OGURA, T. NISHINO and S. SETO

of magnesium chloride, 25 mpmoles of 14C-isopentenyl of 5-11% silicon oil DC-705 (Dow Corning Co.) on pyrophosphate (1.2 ƒÊCi per ƒÊmole) and 10-100 jug of base-washed 30-50 mesh firebrick was used for analysis enzyme protein. After the mixture had been incubated of C10-C15 alcohols. at 37•Ž for 40 min, the reaction was stopped by the For determination of optical densities a Cary addition of 0.2ml of 1 N hydrochloric acid , and the Recording spectrophotometer model 14 was used. mixture was kept at 37•Ž for 15 min to complete the

hydrolysis of acid-labile dimethylallyl pyrophosphate RESULTS formed*. The mixture was made alkaline by addition Enzyme Purification-All steps were carried of a mixture of 0.3ml of 1 N sodium hydroxide and out at 4•Ž unless otherwise stated. A pumpkin 2.0ml of 2M sodium chloride . The mixture was extracted with 5ml of ethyl ether. After washing with (1 kg) was sliced, seeds being removed, pul water an aliquot (1.0ml)** of the extract was assayed verized in a homogenizer (Fuji juicer), and for radioactivity in toluene scintillator without evapo the homogenate was suspended in 500ml of ration of the solvent. For determination of radio 0.1 M phosphate buffer, pH 7.0. The mixture activity a Kobekogyo liquid scintillation counter model was squeezed through gauze, and the filtrate

GSL-111 was used. was centrifuged at 40,000•~g for 1 hr. The Assay for Prenyllransferase (Farnesyl Pyrophosphate supernatant (670ml) was fractionated with Synthetase) Activity-The reaction tubes contained the ammonium sulfate, and the precipitated pro same mixture as that for the assay of isopentenyl tein between 40-65% saturation was dissolved pyrophosphate isomerase activity except that 25 mp moles of non-labeled dimethylallyl pyrophosphate or in a minimum volume of 0.05 M phosphate geranyl pyrophosphate was added. The activity was buffer. Both isomerase and prenyltransferase measured by the same method as that for the iso activities were contained in this 40-65% am pentenyl pyrophosphate isomerase activity except monium sulfate precipitated fraction. Table that ethyl ether was replaced by petroleum ether for I shows the enzymatic conversion of 14C-iso the extraction of radioactive materials. pentenyl pyrophosphate into acid-labile prenyl Thin Layer and Gas Chromatography-Thin layer pyrophosphates catalyzed by the crude fraction chromatography was performed in the two systems without any allylic pyrophosphate added. The described by McSWEENEY (16). In one system (nor resulting solution was placed on a column of mal phase) a plate coated with silica gel which had Sephadex G 200 which had been equilibrated been activated at 140•Ž for at least 1 hr , was deve with 0.01 M phosphate buffer, pH 6 .8, and loped with benzene: ethyl acetate (80:20) . In the eluted with the same buffer other method (reverse phase) a plate coated with . The effluent was Kieselgur G was activated at 110•Ž for 1 hr and then collected in 2ml portions, and each fraction impregnated with paraffin oil. After drying at 110•Ž for a few min, the plate was developed with acetone: TABLE I water (65:35) saturated with paraffin oil . alcohols for reference were located by exposing the Conversion of 14C-isopentenyl pyrophosphate developed plate to iodine vapor. After the plate was into acid-labile material. allowed to stand until the spots disappeared Each incubation mixture contained , sections , in a final of chromatographic plate were then scraped into volume of 1.0ml, 25 ƒÊoles of phosphate buffer , counting vials and the radioactive regions were deter pH 7.0 and 25 mƒÊmoles of 14C-isopentenyl pyro mined by liquid scintillation counting. Gas chro phosphate in addition to MgCl2 and enzyme as matography was carried out with a Hitachi gas indicated. Conditions of incubation and assay chromatograph type KGL-2A. A 2 m column of were the same as described in the section of assay

butanediol succinate on Chromosorb W (Nikkaseiko Co .) for prenyltransferase activity . was used for analysis of C5 alcohols, and a 2m column

* 14C-Isopentenyl pyrophosphate is so stabl e against this mild treatment with acid that it does not give any 14C-material extractable with ethyl ether or petroleum ether. ** The presence of 1 .0ml of ethyl ether or petro leum ether in toluene scintillator had very little quenching effect. Prenyltransferase and Isopentenyl-PP Isomerase 199

was assayed for both isopentenyl pyrophosphate The isomerase fractions (No. 12-17) and the isomerase and prenyltransferase activities. prenyltransferase fractions (No. 20-24) were Concentration of protein was followed by the combined respectively and they were used for measurement of optical densities at 280 mp. most studies. Prenyltransferase emerged a little earlier than Identificationof Products-Figure two shows isopentenyl pyrophosphate isomerase, but the the gas chromatographic separation of alcohols separation was not complete. The fractions liberated by acid treatment of the reaction containing the major part of both enzyme product formed by the incubation of 14C-iso activities were combined, and the combined pentenyl pyrophosphate with the isomerase fraction containing about 500 mg of protein fraction. The radioactivity trapped in the was chromatographed on a column of hydro effluent was present in carrier dimethylvinyl xylapatite (1.6•~16 cm) by elution with a linear carbinol and in carrier dimethylallyl alcohol,

gradient of phosphate buffer, pH 6.8. The indicating the enzymatic formation of di mixing flask contained 150 ml of 0.01 M phos methylallyl pyrophosphate. It is well known

phate buffer, pH 6.8, and the reservoir con that dimethylallyl pyrophosphate is readily tained 150 ml of 0.25 M phosphate buffer, pH hydrolyzed by the treatment with acid to yield 6.8. As seen in Fig. 1, prenyltransferase was a mixture of two isomeric alcohols, and that separated almost completely from isopentenyl the formation of the tertiary alcohol is pre

pyrophosphate isomerase. The curve for the isomerase activity showed two peaks at fraction

9 and 14, but the analysis of the products by both fractions indicated that these two fractions catalyzed the isomerization of isopentenyl

pyrophosphate to dimethylallyl pyrophosphate.

Fio. 2. Gas chromatographic separation of alcohols liberated on acid treatment of the product of the enzymatic reaction. The ethyl ether extract obtained from the reaction mixture of 14C-isopentenyl pyrophosphate with the isomerase fraction by the same method as described in the assay for isopentenyl pyro

phosphate isomerase activity was dried over sodium sulfate for 5 hr. After dimethylvinylcarbinol, dimethylallyl alcohol and isopentenol were added as carriers to the extract, the solution was con centrated to a small volume and was applied on FIG. 1. Chromatographic separation of iso a 2 m column of 20% butanediol succinate. The pentenyl pyrophosphate isomerase and prenyltrans chromatography was carried out at 80•Ž under a ferase on hydroxylapatite. helium gas pressure of 0.15 kg/cm2. Each section Aliquots (0.1 ml) were assayed for enzyme between the points indicated by arrows was trapped activity. in a glass U-tube cooled by dry ice-acetone. The

•\•œ•\ Optical density at 280mƒÊ. tube was washed with toluene scintillator into a counting vial, and the radioactivity was deter •\•›•\ Isopentenyl pyrophosphate isomerase acti

vity. mined. The height of the cross-hatched area indicates the radioactivity of the section. •\• •\ Prenyltransferase activity. 200 K . OGURA, T. NISHINO and S. SETO dominant (17). The absence of radioactivity aration. The formation of dimethylallyl pyro in carrier isopentenol excludes the presence phosphate is suggested by the results given in of phosphatase activity in this enzyme prep Table ‡U. The increase of radioactive material

extractable with petroleum ether in Exp. 3 TABLE ‡U indicates that the isomerase affords dimethyl

Combined incubation of isomerase allyl pyrophosphate necessary for the prenyl and transferase fractions. transferase reaction. Each incubation mixture contained, in a final In order to identify the acid-labile prenyl volume of 1.0ml, 25 ƒÊmoles of phosphate buffer, pyrophosphates formed by the prenyltrans pH 7.0, 5ƒÊmoles of MgCl2 , 25 mtlmoles of 14C ferase fraction the products were treated with -isopentenyl pyrophosphate and enzyme fractions alkaline phosphatase, and the hydrolysates indicated. extracted with benzene were analyzed by means of thin layer and gas chromatographies. The results (Figs. 3 and 5) show that the phos phatase treatment of the products obtained by the reaction of 14C-isopentenyl pyrophosphate with geranyl pyrophosphate in the presence

1) A tenthml of fraction 15 and 23 obtained in the hydroxylapatite chromatography (Fig. 1) was used.

FIG. 4. Gas chromatographic separation of alcohols liberated on alkaline phosphatase treat ment of the product of the enzymatic reaction . To a reaction mixture obtained from the complete incubation of prenyltransferase with 14C- isopentenyl pyrophosphate and dimethylallyl pyro

phosphate, a few milligrams of alkaline phospha tase were added. After standing at room tem

perature overnight, the mixture was made alkaline by addition of 0.2ml of IN sodium hydroxide FIG. 3. Normal phase thin layer chromato and 2ml of 1m sodium chloride . Geraniol and graphy of alcohols liberated on alkaline phosphatase trans-trans farnesol were added as carriers to the treatment of the product of the enzymatic reaction . mixture, and the mixture was extracted with Upper portion, Products obtained when 14C benzene. The benzene solution , after being dried -isopentenyl pyrophosphate and geranyl pyrophos over sodium sulfate and concentrated to a small phate were substrates. Lower portion, Products volume, was applied on a 2 m column of 5% silicon obtained when 14C-isopentenyl pyrophosphate and oil DC 705. The chromatography was carried dimethylallyl pyrophosphate were substrates . Mid out at 178•Ž under a herium pressure of 0 .21 dle portion, Spots of reference alcohols: A , iso kg/cm2. The method of collection of the effluent pentenol ; B, geraniol ; C, trans-trans farnesol ; D, and the determination of the radioactivity were cis-trans farnesol. the same as given under Fig. 2. Prenyltransferase and Isopentenyl-PP Isomerase 201

of transferase gives 14C-farnesol as a single active peak near the solvent front in the product. When 14C-isopentenyl pyrophosphate reverse phase thin layer chromatography (Fig. and dimethvlallyl pyrophosphate were sub 6) is attributable to radioactive isopentenol strates, 14C-farnesol and 14C-geraniol were liberated from unreacted isopentenyl pyro found in the hydrolysates (Figs. 3, 4). The phosphate. The thin layer chromatography enzymatically formed farnesol was a trans-trans of the hydrolysate of the products obtained isomer and neither sis-trans isomer nor nerol by the incubation of 14C-isopentenyl pyro

(cis isomer of geraniol) was detected in the phosphate with crude extract (1000•~g super hydrolysates (Figs. 4, 5). The reverse phase natant of the homogenate) suggests the forma thin layer chromatography (Fig. 6) indicates tion of geranylgeraniol and other non-polar the absence of geranylgeraniol. The radio materials (Fig. 7). Therefore, it is considered

FIG. 7. Reverse phase thin layer chromate FIG. 5. Gas chromatography of farnesol lib erated on alkaline phosphatase treatment of the graphy of hydrolysates of the products obtained by the incubation of 14C-isopentenyl pyrophosphate product obtained by the incubation of prenyl with crude extract. transferase with 14C-isopentenyl pyrophosphate and Incubation was made in the same condition geranyl pyrophosphate. as that described in the section of assay for The sample for the chromatography was pre prenyltransferase. Alkaline phosphatase was used pared in the same way as described under Fig. for the hydrolysis of the products. Spots of 4. The chromatography was performed at 186•Ž reference alcohols: A, geranyllinalool; B, geranyl on a 2 m column of 11% silicon DC 705 on base geraniol; C, farnesol; D, geraniol and isopentenol. washed firebrick with a helium pressure of 0.4 kg/cm2.

FIG. 6. Reverse phase thin layer chromato graphy of alcohols liberated on alkaline phospha tase treatment of the product of the enzymatic Fm. 8. The effect of incubation time on the reaction. formation of acid-labile prenyl pyrophosphates. 14C-Isopentenyl pyrophosphate and dimethyl Spots of reference alcohols: A, geranyllinal ool; B, geranylgeraniol; C, farnesol; D, geranial allyl pyrophosphate were incubated with prenyl and isopentenol. transferase. 202 K. OGURA, T. NISHINO and S. SETO

FIG. 11. The effect of metal ion concentra tion on prenyltransferase activity. FIG. 9. The effect of protein concentration Each reaction mixture contained 25 mƒÊmoles on farnesyl pyrophosphate synthesizing activity. 14C-Isopentenyl pyrophosphate and geranyl of 14C-isopentenyl pyrophosphate, 25 mƒÊmoles of

pyrophosphate were incubated with prenyltrans geranyl pyrophosphate, 40 ƒÊg of enzyme protein, ferase. and MgCl2 (-•œ-) or MnCl2 (-•›-) as indicated.

TABLE ‡V Inhibitory effects by iodoacetamide.

The inhibitor was preincubated with enzyme for 10 min before substrates were added. Each reaction mixture contained about 50 ƒÊg of enzyme protein.

FIG. 10. The effect of geranyl pyrophosphate concentration on prenyltransferase activity. with time of incubation at least up to 120 min Each reaction mixture contained 70 ƒÊg of (Fig. 8). The transferase activity increased enzyme protein, 50 ƒÊmoles of phosphate buffer, with protein concentration (Fig. 9). K value pH 7.0, 5 ƒÊmoles of MgCl2, 25 mƒÊmoles of 14C -isopentenyl pyrophosphate, and geranyl pyrophos for geranyl pyrophosphate calculated from Lineweaver-Burk plot (Fig. 10) is 1.3•~l0-6M. phate as indicated. The mixture was incubated at 37•Ž for 20 min. The velocity of the reaction The pH optimum for transferase activity is is expressed in millimicromoles of farnesyl pyro around 7.5. The prenyltransferase reaction

phosphate formed per minute. requires Mg" or Mull (Fig. 11). Mg++ is a

better activator than Mn++. A partial inhi that the transferase synthesizing farnesyl pyro bition of transferase activity by iodoacetamide phosphate was made free of the geranyl-geranyl at 5•~10-3 M was observed, while isopentenyl

pyrophosphate synthesizing system through pyrophosphate isomerase was completely in this procedure of purification. hibited at 5•~10-3M (Table ‡V). Properties of Pienvltransferase-The conver sion of isopentenyl pyrophosphate into acid DISCUSSIONS labile prenyl pyrophosphate increased linearly Prenyltransferase obtained from pumpkin Prenyltransferase and Isopentenyl-PP Isomerase 203

TABLE ‡W phates and they have shown that dimethylallyl, Prenyltransferase activity with various prenyl geranyl, farnesyl, and geranylgeranyl pyro pyrophosphate. phosphates can be substrates for the condensa tion with isopentenyl pyrophosphate catalyzed by this enzyme preparation. Such a consecu tive reaction specificity of the prenyltransferase seems to be common to all the enzymes related to the synthesis of prenylpyrophosphate. It would be of interest to study what regulates the length of a final product in the consecutive condensation.

The authors are indebted to Takasago Perfumary Co. Ltd., and to Dr. Isler of Hoffmann-La Roche and Company for their kind supply of authentic 1) Abbreviation used : PP, pyrophosphate ester samples of terpene alcohols. fruit catalyzes the condensation reactions of REFERENCES isopentenyl pyrophosphate with dimethylallyl (1) F. Lynen, B.W. Agranoff, H. Eggerer, U. Henning, and E.M. Moslein, Angew. Chem., 71, pyrophosphate as well as with geranyl pyro 657 (1959) phosphate to yield trans-trans farnesyl pyro (2) C.R. Benedict, J. Kett, and J.W. Porter, Arch. phosphate as a final product. This function Biochem. Biophys., 110, 611 (1965) of catalysis was the same as farnesyl pyro (3) J.K. Dorsey, J.A. Dorsey, and J.W. Porter, J. phosphate synthetase of pig liver by PORTER Biol. Chem., 241, 5353 (1966) et al. (3) and by HOLLOWAY and POPJAK (4). (4) P.W. Holloway and G. Popjak, Biochem. J., 104, The properties of the enzyme are also similar 57 (1967) to those of the pig liver enzyme. Neither (5) De W.S. Goodman and G. Projak, J. Lipid Res., geranylgeranyl pyrophosphate synthetase nor 1, 286 (1960) isopentenyl pyrophosphate isomerase activity (6) L.A. Witting and J.W. Porter, J. Biol. Chem., was present in this preparations. Table ‡W 234, 2841 (1959) shows the function of the transferase. No (7) B.W. Agranoff, H. Eggerer, U. Henning, and F. Lvnen, 1. Biol. Chem., 235, 326 (1959) transformation of farnesyl residue accounts (8) H.S. Damayanti, W.W. Cleland, and J.W. Porter, for the absence of geranylgeranyl or any J. Biol. Chem., 240, 1946 (1965) longer chain prenyl pyrophosphates in the (9) P.W. Holloway and G. Popjak, Biochem. J., 104, reaction product. 25P (1967) Repeated chromatography of the trans (10) D.L. Nandi and J.W. Porter, Arch. Biochem. ferase fraction on hydroxylapatite with an Biophys., 105, 7 (1964) alternate elution resulted in the recovery of (11) A. Tiselius, S. Hjerten, and 0. Levin, Arch. the enzyme protein in a single peak, and Biochem. Biophys., 65, 132 (1956) relative enzyme activity with dimethylallyl (12) C. Yuan and K. Bloch, J. Biol. Chem., 234, 2605 and with geranyl pyrophosphates did not (1959) change. KANDUTSCFet al. (13) reported the (13) A.A. Kandutsch, H. Poulus, E. Levin, and K. Bloch, J. Biol. Chem., 239, 2507 (1964) isolation of geranylgeranyl pyrophosphate (14) K. Bloch, S. Chaykin, A.H. Phillips, and A.De synthetase from Micrococcuslysodeikticus which Waard, J. Biol. Chem., 234, 2595 (1959) catalyzes the condensations of isopentenyl (15) K. Shishido and H. Nozaki, " Yukigoseiho ", pyrophosphate with dimethylallyl, geranyl, and Gihodo, Tokyo, Vol. VI, p. 51 (1957) farnesyl pyrophosphates to give geranylgeranyl (16) G.P. McSweeney, J. Chromatog., 17, 183 (1965) pyrophosphate as a major product. More (17) W.R. Gottingen, Angew.Chem. (Internatl. Edit.), recently, ALLENet al. (18) obtained an enzyme 4, 444 (1965) system from the same organism which catalyzes (18) C.M. Allen, W. Alworth, A. Macrae, and K. the formation of long chain prenyl pyrophos Bloch, J. Biol. Chem., 242, 1895 (1967)