The Works of Several Investigators Have Shown Mevalonic Acid

The Journal of Biochemistry, Vol. 52, No. 6, 1962

5-Phosphomevalonic Acid as the Intermediate of the Enzymatic Synthesis of Bacterial Phytoene

By SHIRO OHNOKI, GINZABURO SUZUE and SHOZO TANAKA

(From the Department of Chemistry, Faculty of Science, Kyoto University, Kyoto)

(Received for publication, July 10, 1962)

The works of several investigators have of the Formosan cobra (Naja naja atra), a yellowish shown mevalonic acid (3, 5-dihydroxy-3-me- amorphous material, was a generous gift from Prof. T. thylvaleric acid, MVA) to be one of the Suzuki of Kyoto University. Adenosinetriphosphate efficient precursors of carotenoids (1-8). One (ATP) equally labeled with P32 at the ƒÀ and ƒÁ posi of the present authors has also succeeded in tions was synthesized by condensation of adenosine 5•Œ-monophosphate and pyrophosphate with N, N•Œ the enzymatic synthesis of bacterial phytoene dicyclohexylcarbodiimide (12). P32-Phosphoric acid from 2-C14-mevalonic acid by a phytoene- was a gift from the Japan Atomic Research Institute, accumulating mutant of Staphylococcus (9,10). Tokai. Other reagents were the same as reported The phosphorylated intermediates are known in previous papers (9, 10).

in case of the biosynthesis of steroids (11), Radioactivity was measured as infinitely thin but no detail has been reported in the case of samples in a gas-flow counter, Model D-47, of carotenogenesis. Nuclear Chicago Corp., U. S. A. Radioactivity dis This paper is concerned with the identi tributions on filter papers after paper chromatography fication of 5-phosphomevalonic acid (5-P- or high-voltage paper electrophoresis were determin MVA) among the intermediary products in ed with a set of automatically scanning and record- ing instruments in a gas-flow system, Actigraph of phytoene biosynthesis from mevalonic acid, Nuclear Chicago Corp, U. S. A., Model 1620 B, C-

and its enzymatic conversion into phytoene 100 B, R 1000 and D-47.

with more than twice the efficiency of that Paper Chromatography-For paper chromatography,

obtained with DL-mevalonic acid. The pre the following five solvent systems were used by the sence of 5-pyrophosphomevalonate (5-PP- ascending method at 25°C with Toyo-roshi No. 50: MVA) as an intermediate was detected by n-butanol-formic acid-water (77: 10: 13, by volume), paper chromatography. The biosynthetic methanol-conc. ammonia-water (60: 10: 30), iso steps in carotenoid formation are believed to butanol-formic acid-water (40: 10: 16) and tert be identical with those of steroid biosynthesis, amylalcohol-acetic acid-water (4: 1 : 2). High-voltage Paper Electrophoresis-For study with at least in the early stages. high-voltage paper electrophoresis, an apparatus of

high-voltage electrophoresis of Shirai-matsu Manufac

turing Co., Osaka, type D-2, was used. Determination of the Ratio of P32 and C14-The

doubly labeling experiment with C14 and P32 was

performed with DL-2-C14-mevalonic acid and ATP32

MATERIALS AND METHOD (ƒÀ, ƒÁ position labeled). The purified product was Chemicals and Instruments-2-C14-Mevalonie acid counted with and without an aluminum window. and mevalonic acid lactone used were purchased The counting efficiencies with the aluminum window from the Radiochemical Centre, Amersham (England), used were predetermined respectively for standard and Fluka A. G. Chemische Fabrik, Buchs SG samples of P32 and C14. (Switzerland), respectively. They were converted to Preparation of Enzyme-The method of preparation the potassium salts by neutralization of their aqueous of the cell-free enzyme from phytoene-accumulating solutions with a small excess of potassium hydroxide mutant of Staphylococcus was the same as described and incubation at 37°C for 30 minutes. The venom previously (9, 10). The enzyme preparation contain- 423 424 S. OHNOKI, G. SUZUE and S. TANAKA

ed 20-35 mg. of protein per ml. The protein con- tent of preparation was measured by the Folin- Ciocalteu reagent (13) using egg-albumin (E. Merk, Darmstadt, Germany) as standard. The extracted enzyme lost biosynthetic activity for the production of phytoene from mevalonic acid in less than 3 days, even when it was stored in the frozen state at -20°C. FIG. 1. Chromatogram of derivatives from 2- Mevalonic kinase activity was stable for more than C14-mevalonic acid after the development of iso several weeks in frozen state. butyric acid-conc. ammonia-water (66: 3: 30).

EXPERIMENTAL AND RESULTS acid, respectively, reported by de W a a r d and Detection of Mevalonic Acid Derivatives Form- P o p j a k (14). The spot Z was probably ed during Short Term Incubation-The enzymatic a polymerized pyrophosphate(s) synthesized formation of at least three new derivatives from 2-C14-mevalonic acid. of mevalonic acid was observed in short term Preparation of Substance X (5-Phosphomeva- (5-10 minutes, at 37°C) incubations with the lonic Acid)-A small scale of preparation of X `complete system' for phytoene synthesis from was performed in the following simple system : mevalonic acid. The complete system con ATP (5 ƒÊmoles), tris buffer (pH 8.0, 20 ƒÊmoles), tained triphosphopyridine nucleotide (TPN ; MnSO4 (5 ƒÊmoles), KF (5 ƒÊmoles), enzyme

0.5 mg.), 6-phosphogluconate (2 ƒÊmoles), nico (0.15 ml.) and DL-2-C14-mevalonic acid (0.02 tinamide (50ƒÊmoles), ATP (10 ƒÊmoles), flavine ƒÊ mole, 2.2 x 104 c.p.m.) in a volume of 0.21ml. ; adenine dinucleotide (FAD ; 0.05 mg.), tris which was incubated for 3 minutes at 37°C. buffer (pH 8.0, 40 ƒÊmoles), MnSO4 (10 ƒÊmoles), For the purpose of identification of the MgSO4 (5 ƒÊmoles), Tween 20 (final concentra substance X, a larger scale of incubation

tion; 0.17 per cent), enzyme (20-35mg. of (equivalent to 30-50 times as much as in the protein per ml.; 0.30 ml.), KF (10 ƒÊmoles) and small scale experiment) was possible without DL-2-C14-mevalonic acid (0.02 ƒÊmole, 2.2 x 104 the purification of mevalonic kinase. After c.p.m.) in a volume of 0.6 ml. After incuba incubation, the reaction mixture was depro tion for 5 minutes at 37°C, the reaction was teinized by centrifugation at 0°C and freed stopped by heating for 3 minutes at 100°C. from nucleotides and mevalonate by paper Paper chromatography of such a sample with chromatography with tert-butanol-formic acid- the iso-butyric acid-conc. ammonia-water water system. Compound X was eluted with system showed the presence of three new water and chromatographed at least three radioactive substances ; X (R f : 0.37-0.43), Y more times on paper with methanol-conc.

(0.29-0.33) and Z (0.12-0.21). ammonia-water system to be free from inor

Further experiments showed that the three ganic phosphate. derivatives of mevalonic acid were also form- The final yield of pure X was 15-30 per ed even in a 'simple system' containing ATP cent judging from its radioactivity.

(10 ƒÊmoles), tris buffer (pH 8.0, 40 ƒÊmoles), Rf Values of X by Paper Chromatography- MnSO4 (10 ƒÊmoles), KF (10 ƒÊmoles), DL-2-C14- R f values of compound X in five solvent mevalonic acid (0.02 ƒÊmole, 2.2 x 104 c.p.m.) systems are given in Table I. Upon com and enzyme (0.2 ml.) in a total volume of parison with R f data reported in the previous 0.42 ml. papers for the authentic substance (13-19), A typical chromatogram is shown in compound X was identical as 5-phospho- Fig. 1. mevalonic acid. The two least polar substances (Rf : 0.75 Paper Electrophoresis-The compound X and 0.66) were mevalonic lactone and me and mevalonic acid migrated 21.6 cm. and valonic acid respectively. The Rf values of 16.8 cm. respectively under high-voltage paper X and Y coincided with that of 5-phospho- electrophoresis : pyridine-acetate buffer (pH mevalonic acid and 5-diphosphomevalonic 6.2), 41 volts/cm., 65 mA, 120 minutes (paper: 5-Phosphomevalonate : Intermediate of Phytoene 425

TABLE I Deternination of Ratio P" to C14-The Rf Values of Compound X doubly labeled compound X with C14 and P32 was enzymatically synthesized using DL-2-C14- mevalonic acid and ATP32 with a simple system, and it was purified by repeated paper chromatography as described before. The product was adsorbed on Dowex-l formate column (12 x 0.8 cm.) and gradient elution was performed, The compound was eluted with 3.5 N to 4.0 N formic acid as described by Pure X enzymatically synthesized from 2-C14- B l o c h et al. (18). mevalonic acid was paper chromatographed As shown in Table II, the ratio of P32 to (ascending) for the following five solvent systems. C14 in compound X contains mevalonic acid The component of each system : A, n-butanol- and phosphate in the mole ratio of 1 to 1. formic acid-water (77: 10: 13, by volume) ; B, methanol-conc. ammonia-water (60: 10: 30); C, TABLE II iso-butyric acid-conc. ammonia-water (66: 3 : 30) ; D, tert-butanol-formic acid-water (40: 10: 16) ; E, Ratio of P32 to C14 of Doubly Labeled X tert-amylaleohol-acetic acid-water (4: 1 : 2).

Toyo-roshi, No. 2). This result shows that compound X behaves the same as 5-phosphomevalonic acid according to data previously ATP32 (20 ƒÊmoles ; specific activity: 4.5 X 10° reported (19). c.p.m./ƒÊmole in terminal P atom), MnSO4 (20 Detection of Phosphorus-The presence of ƒÊ moles), KF(20ƒÊmoles), enzyme (protein concent phosphorus was confirmed by the molybdate ration) : 24 mg./ml. ; 0.5ml.) and DL-2-C14-me- reagent of Hanes and Isherwood (20). valonic acid (1 ƒÊmole ; specific activity : 1.0 X 105

A slightly blue spot was observed upon spray- c.p.m./ƒÊmole) in a volume of 1.0 ml. were used. ing the substance X (0.3ƒÊmole equivalent) Incubation 5 minutes at 37°C. with the reagent and gentle heating. The incorporation of radioactivity of ATP32 into Stability to Acid and Alkali-The stability compound X also confirmed the presence of of compound X to acid, alkali and heat treatment was investigated. After each treat phosphorus. Enzymatic Hydrolysis of Compound X-Com- ment, the reaction mixture was chromate-

pound X (0.16ƒÊmole equivalent) was incubated graphed on paper with n-butanol-formic acid- for 25 hours at 37°C in the system containing water system and the distribution of radioac tivity was determined by actigraphic analysis. glycine buffer (pH 8.5, 10ƒÊmoles), MgCl2 (1 ƒÊ mole) and amorphous phosphatase of snake The results are summarized as shown in venom (1 mg.) in a volume of 0.11 ml. After Table III. the reaction was stopped by the addition of Test for Absence of Lactone Ring-The filter- trichloroacetic acid (final concentration : 5 per ed solution of an equivolume mixture of 7% cent), the incubation mixture was spotted on hydroxamine hydrochloride in methanol and 7.2% potassium hydroxide in methanol was paper and developed with the n-butanol formic acid-water system. The result of acti sprayed on spots of mevalonate (0.1 ƒÊmole) and compound X (0.1 ƒÊmole equivalent) on graphic analysis showed that mevalonic acid filter paper. This was followed by a spray (R f : 0.85) was regenerated in 70% yield. of 1 % ferric chloride in 1% hydrochloric acid Spraying the paper with the molybdate rea solution. Only mevalonate showed a reddish gent produced a yellow spot (Rf : 0.13) to orange color within 15 minutes. The failure show orthophosphate was liberated in the of compound X to yield a positive test clearly reaction. 426 S. OHNOKI, G. SUZUE and S. TANAKA

TABLE III aliquots of the petroleum benzin, containing Stability Test of Compound X the unsaponifiable fraction, were dried on aluminum planchets and the radioactivity were counted as infinitely thin samples. Separation of the carotenoid in the unsaponi fiable fraction were carried out by chromato

graphy on an activated-alumina column. The fractions containing purified phytoene were dried on aluminum planchets and radioactivity were measured as described before. Stability of compound X were examined These procedures were described in detail in under conditionsas shownin table. Values are previous papers (9, 10). The results are sum expressed, after the paper chromatographyof n marized in Table ‡W. butanol-formicacid-water system, with the result

of actigraphic analysis. DISCUSSION showed the absence of a lactone ring in this Compound X which is formed in the compound. first step in the synthesis of phytoene from Enzymatic Synthesisof Bacterial Phytoenefrom mevalonate, is also produced in the simple 2-C14-5-PhosphomevalonicAcid-2-C14-5-Phospho- system. This compound showed properties mevalonic acid and DL-2-C14-mevalonicacid identical with those of 5-phosphomevalonic were incubated in test tubes at 37°C for 3 acid upon paper chromatography, by column hours with gentle shaking in the complete chromatography and by paper electrophoresis. system as described previously. The enzy This compound was concluded to be a mono matic reactions were stopped by the addition phosphorylated derivative of mevalonic acid of 2 ml. of 10 per cent of ethanolic sodium by the isolation of mevalonic acid after treat hydroxide to the mixture ; and 10 mg. of p ment with phosphatase. It contained one hydroquinone and 1.00 ml. of a petroleum atom of phosphorus per mevalonic acid unit. benzin solution of pure bacterial phytoene of The phosphorus is probably present in the absorbancy 9.37 at 286 m t were also added compound as a phosphate ester as it proved simultaneously. After saponification, the to be stable to hydrolysis for 7 minutes in N HCl or N NaOH at 100°C. Its failure to TABLE ‡W give a lactone test showed that esterification

Biosynthesis of Phytoene from Mevalonate and is probably at the 5-hydroxy group. Judging

5-Phosphomevalonate from these characteristics, compound X is undoubtedly 5-phosphomevalonic acid, a substance which was isolated by T c h e n (15) and other investigators (14, 16-19) in studies on squalene, cholesterol and terpene biosyn theses. Compound X was incorporated into phytoene twice as efficiently as DL-2-C14-mevalonate in the complete system. Therefore , Each system contained TPN (0.5 mg,), 6 the carotenoid phytoene is probably synthesiz - phosphogluconate (2ƒÊmoles), nicotinamide (50 ed through steps quite similar, at least in the ƒÊ moles), ATP (10ƒÊmoles), FAD (0 .05 mg.), Iris buffer (pH 8.0, 40ƒÊmoles), MnSO4 (10ƒÊmoles) initial stage, as in other isoprenoid com- , enzyme (protein concentration, 33 mg./ml.) and pounds. the substrate indicated, in a volume of 0,6 ml.

Incubation in nitrogen (99.9%) for 3 hours at SUMMARY 37°C. 1. The formation of phosphorylated 5-Phosphomevalonate : Intermediate of Phytoene 427

derivatives of mevalonic acid in the presence (8) Grob, E. C., "Ciba Foundation Symposium on of adenosine triphosphate and Mn++ by the Biosynthesis of Terpenes and Sterols", ed. enzymes extracted from Staphylococcus is de by Wolstenholme, G. E. W., and O'Connor, M., scribed. J. & A. Churchill Ltd., London, p. 267 (1959) 2. One of the products was purified by (9) Suzue, G., Biochim. et Biophys. Acta, 45, 616 (1960) various chromatographic procedures and (10) Suzue, G., J. Biochem., 51, 246 (1962) shown to be identical with 5-phosphomeva- (11) Popjak, G., and Cornforth, J. W., Advances in Enzymol., 22, 281 (1960) lonic acid. (12) Lowenstein, J. M., and Metzenberg, R. L., 3. 5-Phosphomevalonic acid was shown " Biochemical Preparations " ed , by Lardy, H. to be converted to phytoene with twice the A., Academic Press Inc., New York, Vol. VII efficiency of the DL-mevalonic acid. p. 5 (1960) (13) Lowry, O. H., Rosebrough, N. J., Farr, A. L., REFERENCES and Randall, H. J., J. Biol. Chem., 193, 265 (1) Braithwaite, G.D., and Goodwin, T.W. Biochem. (1951) J., 67, 13 p (1957) (14) de Waard, A., and Popjak, G., Biochem. J., 73, (2) Goodwin, T. W., Biochem. J., 70, 612 (1958) 410 (1959) (3) Braithwaite, G. D., and Goodwin, T. W., Bio (15) Tchen, T. T., J. Biol. Chem., 233, 1100 (1958) chem. J., 76, 5 (1960) (16) Markly, K., and Smallman, E., Biochim. et Bio (4) Braithwaite, G. D., and Goodwin, T. W., Bio phys. Acta, 47, 327 (1961) chm. J., 76, 194 (1960) (17) Chaykin, S., Law, J., Phillips, A. H., Tchen, (5) Purcell, A. E., Thompson, Jr. C. A., and Bon T. and Bloch, K., Proc. Natl. Acad. Sci., 44, ner, J., J. Biol. Chem., 234, 1081 (1959) 998 (1958) (6) Chichester, C. 0., Yokoyama, H., Nakayama, (18) Bloch, K., Chaykin, S., Phillips, A. H., and de T. O. M., Lukto, A., and Mackinney, G., J. Waard, A., J. Biol. Chem., 234, 2595 (1959) Biol. Chem., 234, 598 (1959) (19) Henning, U., Moslein, E. M., and Lynen, F., (7) Anderson, D. G., Norgard, D. W., and Porter, Arch. Biochem. Biophys., 83, 259 (1959) J. W., Biochem. Biophys. Research Communns., 1, (20) Hanes, C. S., and Isherwood, F. A., Nature, 164, 83 (1959) 1107 (1949)