[2-C14] Mevalonic Acid by This Phytoene-Ac Cumulating Mutant Was Investigated (4) and of the Composition As Shown in Table I

The Journalof Biochemistry,Vol. 51, No. 4, 1962

On the Enzymatic Synthesis of Bacterial Phytoene from [2-C14] Mevalonic Acid

By GINZABUROSUZUE (Fromthe Departmentof Chemistry,Faculty of Science,Kyoto University, Kyoto) (Receivedfor publication,November 13, 1961)

It is already known that although a concentrated H2SO4, water, and 2% solution of NaOH, and was dehydrated over CaCl2. Ether used was normal strain of Staphylococcus aureus 209P

accumulates predominantly two bright-colored previously treated with p-hydroquinone to remove peroxides. Carrier phytoene was extracted from the pigments, ƒÂ-carotene and rubixanthin (1, 2), mutant cells of Staphylococcus as described later. the culture alwasy becomes colorless (1) when Radioactivity was measured as infinitely thin the bacteria have once developed a high samples in a gas-flow counter, Model D-47, of Nuclear resistance to more than 300ƒÊg. per ml. of Chicago Corp., U.S.A. Ultraviolet absorption was tetracyclines. A mutant, which was found to measured by a Hitachi Recording Spectrophotometer accumulate the carotenoid, phytoene, was Model EPS-2. separated from the colorless resistant mutants Test Organism and Cultivation•\A mutant of

(3). Enzymatic synthesis of phytoene from Staphylococcus aureus (3), which has been shown to ac cumulate phytoene, was cultured in a synthetic medium [2-C14] mevalonic acid by this phytoene-ac cumulating mutant was investigated (4) and of the composition as shown in Table I. The cells

mevalonic acid was ascertained to be one of grown on an agar slant for one day at 28•Ž were transferred to I liter of the medium shown in Table the direct precursors of phytoene, as in the I and cultured with continuous shaking for 18hours case of other isoprenoid compounds (5-8). at 34•Ž or in some cases, for 24hours at 28•Ž. Such In the biosynthesis of phytoene from an aerobic culture was found to give the optimum mevalonic acid, ATP, TPNH and Mn++ (4) yield of cells and enzymatic activity. were required as cofactors, as in the case of Enzyme Preparation-The cells were harvested by

squalene (9-11). The present work showed centrifugation at 4,000r.p.m. for 10minutes, washed that FAD is also a required cofactor when an with a 0.3% aqueous solution of NaCl, and were col enzyme preparation partially purified with lected by centrifugation. They were washed with 0.1M

ammonium sulfate is used. Optimum condi Tris buffer (pH 8.0) containing 1% EDTA and again

tions for biosynthesis of phytoene by these centrifuged down. -preparations were investigated using [2 -C14] After grinding the cells with alumina (Wako Pure Chemical Industries, Ltd., W-800) for 10minutes mevalonic acid as a substrate. Some pro under cooling, enzymes were extracted with 0.01M perties of bacterial phytoene are also described. Tris buffer (pH 8.0).

EXPERIMENTAL For the first series of experiments (cf. Table II-V), the supernatant obtained after the removal of alumina Chemicals and Instruments•\The [2-C14] mevalonic and cell debris by centrifugation (5,000r.p.m. for 10 acid used was purchased from the Radiochemical minutes and 8,000r.p.m. for 10minutes) was used as Centre, Amersham, (England), and other reagents the enzyme preparation. For the latter series of ex used were of commercial origin. Petroleum benzin periments (cf. Table VI-IX), the supernatant was ob (b.p. 50-120•Ž or 50-75•Ž) was purified by successive tained after removal of alumina and cell debris by washings with concentrated H2SO4, mixed solution of centrifugation (29,000•~g for 5minutes and 23 concentrated H2SO4 and concentrated HNO3 (3: 1), ,000•~g for 30minutes). The protein was centrifuged down Abbreviations: ATP, adenosine triphosphate; (29,000•~g for 20minutes) after the addition of as much DPN, diphosphopyridine nucleotide; TPN and TPNH, as 9volumes of saturated ammonium sulfate solution oxidized and reduced triphosphopyridine nucleotide; to the supernatant. The precipitated protein obtained FAD, flavine adenine dinucleotide; Tris, tris(hydroxy methyl)aminomethane; EDTA, ethylenediaminetetra from the supernatant was dissolved in the same volume acetaten of 0.1M Tris buffer (pH 8.0), and used for experiments . 246 Enzymatic Synthesis of Bacterial Phytoene 247

TABLE I on aluminum planchets and the radioactivity was Culture Medium Used .for Cultivation of Coccus counted as described before. Purification and Properties of Bacterial Phytoene•\The harvested cells (wet weight, 100g.) were extracted with boiling ethanol (100ml.) and then 3times with

boiling acetone (50ml.•~3). The extracts were com bined and concentrated to 100ml. under reduced

pressure. Two volumes of water were added to the concentrate and this was extracted 3times (40ml.•~ 2, 20ml.•~1) with petroleum benzin. The extracts

Total protein content of the preparation was were combined and saponified by heating for 2hours

measured by the Folin-Ciocalteu reagent (12). with ethanol (150ml.), potassium hydroxide (40g.), The enzyme extract was unstable and was water (40ml.) and p-hydroquinone (0.5g.). difficult to store for more than two days even in a The unsaponified matter was extracted repeated

frozen state at -10•Ž. The freshly prepared extract ly with petroleum benzin (20ml.•~4, 10ml.•~2) and was, therefore, used throughout in these experiments. the extracts were combined, washed with five 300-ml.

Experimental Conditions•\Each reaction mixture was portions of water, and dried over anhydrous sodium incubated in a test tube at 37•Ž for 3hours with sulfate.

gentle shaking (90-110 strokes per minute). Total Further purification of the extract was carried volume of the solution was always made to 0.6ml. out by chromatographic separation on activated

with addition of 40ƒÊ moles of Tris-HCI or ethylene alumina as described before, but on a larger scale. diamine-HCl buffer. Fluoride was added after all After the fractional separation of the eluate, the other ingredients. The incubation was carried out in ultraviolet spectrum of each fraction was checked.

an atmosphere of air or 99.9% nitrogen gas. The phytoene fraction was immediately dried in vacuo

Treatment of Lipids Produced•\The enzymatic reac (final yield, 13mg.). This fraction showed the same tion was stopped by addition of 2ml. of 10% ethanolic ultraviolet spectrum and absorbancy in petroleum sodium hydroxide to the mixtures. Ten mg. of p- benzin solution as phytoene obtained from tomatoes hydroquinone and 1.00ml. of petroleum benzin, in and carrot oil (13).

which pure bacterial phytoene of absorbancy 8.500 at When two or three drops of a hexane solution of 286mƒÊ had been dissolved, were also added simultane this substance (1mg./ml.) were dropped gently onto

ously. The mixture was heated for 15minutes on a concentrated H2SO4,an orange color appeared at the water bath in a nitrogen atmosphere to saponify the interface which subsequently diffused throughout the esters. After saponification, 2ml. of water and 5ml. acid phase. When 250ƒÊg. of this substance was dis

of petroleum benzin were added and the mixture was solved in 0.1ml. of CHCl3 and treated with 0.4ml. shaken vigorously to transfer the unsaponifiable matter of a saturated CHCl3 solution of SbCl3, a light-green

into the petroleum benzin layer. The petroleum benzin color was produced which slowly changed to yellow extract was washed 3times with water and dehydrat and finally to brown. Following addition of two or

ed over anhydrous sodium sulfate. Aliquots of the three drops of concentrated H2SO4, it became scarlet unsaponifiable fraction thus obtained were dried on in color. When 250ƒÊg. of this substance (in 0.1ml.

aluminum planchets and the radioactivity was counted. of CHCl3) was dissolved in 0.5ml. of acetic anhydride

Column Chromatography of Phytoene•\Separation of and treated with one or two drops of concentrated the carotenoid in the unsaponifiable fraction was H2SO4, a transient violet color developed at once. carried out by chromatography on an activated In a few seconds, the color changed successively from

alumina column (150-200 mesh). The dry petroleum blue to gray, to greenish yellow, to yellow-brown, and benzin solution of the unsaponifiable fraction was finally to a stable red-brown color. The infrared absorption spectrum was measured poured into the column of alumina (1cm.•~15cm.). After washing the column with petroleum benzin, the with a Koken DS-301 infrared spectrophotometer

adsorbed carotenoid was eluted with a mixture of (sodium chloride prism) in sodium chloride cells with 0.02mm. lead shims and it closely resembled that of petroleum benzin and ether (95:5) and 3ml. fractions were collected by using a fraction collector. The phytoene as previously described (13). Phytoene from ultraviolet absorption spectrum of every 3ml. of the a mutant of Staphylococcus is considered to be the same eluate was measured. At least 90% recovery of the substance as that obtained from other sources. Pre carrier phytoene was confirmed by these manipula liminary experiments showed that bacterial phytoene tions. The fractions containing phytoene were dried was converted into ƒÂ-carotene by enzymes extracted 248 G. SUZUE

from ƒÂ-carotene-accumulating Staphylococcus (14). There TABLE III fore, phytoene is thought to be a precursor of ƒÂ- Effect of Concentration of Potassium Fluoride and Nicoti carotene. namide upon the Biosynthesis of Phytoene with Crude

RESULTS Enzyme Extracts Potassium fluoride or nicotinamide was added as Effect of Fluoride, Nicotinamide, and/or EDTA indicated. Enzyme solution, 0.3ml. (protein concen •\ The effect of fluoride, nicotinamide, and/ tration, 34.4mg./ml.). Other ingredients and condi or EDTA on the synthesis of phytoene by tions were the same as those described in Table II. these systems was examined. As shown in

Table ‡U, addition of fluoride and nicotinamide

favored the conversion of DL-[2-C14] mevalonic

acid into phytoene. The optimal concentra

tion of fluoride and nicotinamide for the

biosynthesis of phytoene was examined with

crude enzymes and the results shown in Table

III were obtained. Addition of 10ƒÊmoles of

flouride and 50ƒÊmoles of nicotinamide per

TABLE II

Effect of Fluoride, Nicotinamide, and/or EDTA upon

Conversion of DL-[2-C14] Mevalonic Acid into Unsaponifa ble Fraction and Phytoene with Crude Enzyme Extracts

Each system contained Tris buffer (pH 8.0), 40

ƒÊ moles; ATP, 30ƒÊ moles; DPN, 2mg.; TPN, 0.5mg.; cofactors for this system. Boiled extract from 6-phosphogluconate, 2ƒÊmoles; enzyme solution, 0.3 the cells (cf. Table V) was found to have a ml. (protein concentration, 52mg./ml.); DL-[2-C14] stimulating effect. The biosynthesis of phy mevalonic acid, 1.45ƒÊg. (10,000c.p.m.); MgCl2, 10ƒÊ toene by enzyme preparations precipitated moles, and MnSO4, 4ƒÊmoles. The indicated systems contained EDTA, 7ƒÊmoles; nicotinamide, 50ƒÊmoles; with 90% saturated ammoium sulfate solution

and/or KF, 30ƒÊmoles in a total volume of 0.6ml. was found to occur only to a very small ex Incubation in air for 3hours at 37•Ž. tent without the addition of the 'boiled bacterial extract'. Among many cofactors tested, FAD showed a remarkably stimulating effect (cf. Table VI). As shown in Table VII, the optimum concentration of ATP was l0ƒÊmoles per 0.6 ml. and concentration above this caused an inhibition of phytoene synthesis. Effect of Cysteine, Air, and Nitrogen•\As shown in Table VI, addition of cysteine was observed 0.6ml. was found to produce the most favora to be advantageous to the biosynthesis of

ble conditions for this biosynthesis. phytoene only when the incubation was carri Effect of pH•\The effect of PH was ex ed out aerobically. In an atmosphere of amined by using ethylenediamine hydro nitrogen gas (99.9% purity), conversion of chloride (pH6.0-7.2) or Tris-HCl buffer (pH mevalonic acid to unsaponifiable matter and 7.2-8.8) as shown in Table IV. The optimum phytoene was markedly stimulated and, in

pH was found to be pH 8.0 (Tris buffer). this case, the effect of cysteine was almost Preliminary results showed that phosphate unnoticeable (cf. Table IX). buffer was unfavorable for this experiment. Effect of Metals•\The addition of Mg++ Effect of Cofactor Nucleotides•\ATP and and 6-phosphogluconate was found to be the TPNH were both shown to be essential best method for the generation of TPNH Enzymatic Synthesis of Bacterial Phytoene 249

TABLE IV generate TPNH. As shown in Table VIII, Effect of pH upon the Biosynthesis of Phytoene with the effects of the addition of bivalent cations Crude Enzyme Extracts

Ethylenediamine or Tris buffer, 40ƒÊmoles, was TABLE VI used. Enzyme solution, 0.3ml. (protein concentration, Effect of TPN, FAD, Cysteine, and Nitrogen on the

38.6mg./ml.). Other ingredients and conditions were Biosynthesis of Phytoene with Ammonium Sulfate-pre the same as those in Table II. cipitated Enzymes

Each system contained Tris buffer (pH 8.0), 40ƒÊ moles; ATP, 30ƒÊmoles; 6-phosphogluconate, 2ƒÊmoles; DL-[2-C14] mevalonic acid, 3.19ƒÊg. (22,000c.p.m.); MgCl2, 5ƒÊmoles; MnSO4, l0ƒÊmoles; KF, l0ƒÊmoles; nicotinamide, 50ƒÊmoles, and 0.3ml. 0.1M Tris buffer solution of enzymes precipitated with 90% saturated solution of ammonium sulfate (protein concentration, 28.0mg./ml.) in a total volume of 0.6ml. The in dicated system contained TPN, 0.5mg.; FAD, 50ƒÊg., and/or cysteine,10ƒÊmoles. Incubation in air or in nitrogen (99.9%) for 3hours at 37•Ž.

TABLE V Effect of Cofactors upon the Biosynthesis of Phytoene

with Crude Enzyme Extracts

Cemplete system contained the same ingredients

as those given in Table II. Enzyme solution, 0.3ml.

(protein concentration, 52mg./ml.). Incubation in air at 37•Ž for 3hours.

TABLE VII Effect of ATP Concentration upon the Biosynthesis of

Phytoene with Ammonium Sulfate-precipitated Enzymes ATP was added as indicated. Tris buffer (pH 8.0), 40ƒÊmoles; 6-phosphogluconate, 2ƒÊmoles; DL-[2- C14] mevalonic acid, 3.19ƒÊg. (22,000c.p.m.); MgCl2, 5ƒÊmoles; MnSO4, 10ƒÊmoles; TPN, 0.5mg.; FAD, 50

ƒÊ g.; KF, 10ƒÊmoles; nicotinamide, 50ƒÊmoles, and 0.3 ml. 0.1M Tris buffer solution of enzymes precipitated with 90% saturated ammonium sulfate solution (pro tein concentration, 28.0mg./ml.) were used. Incuba tion in nitrogen (99.9%) for 3hours at 37•Ž.

1) Boiled bacterial extract was prepared from the boiled extract of cells by acetone precipitation with

subsequent drying of the precipitates in a vacuum desiccator.

from TPN in this enzyme system as measured

by a spectrophotometric assay at 340mƒÊ.

Throughout this investigation, therefore, Mg++,

6-phosphogluconate and TPN were used to 250 G. SUZUE

TABLE VIII on phytoene bioynthesis in addition to 5ƒÊ

Effect of Metals upon the Biosynthesis of Phytoene with moles of Mg++ (which was essential for the

Ammonium Sulfate-precipitated Enzyme generation of TPNH in these systems) were investigated. Mn++ was the most effective Each system contained ATP, 10ƒÊmoles and 0.3 cation in stimulating this biosynthesis and ml. 0.1M Tris buffer solution of enzymes precipitat ed with 90% saturated ammonium sulfate solution Mg++ was found to be much less effective

(protein concentration 26.0mg./ml.) in a total than Mn++. volume of 0.6ml. Other ingredients and condi Effect of Detergent•\The effect of detergent tions were the same as those given in Table VII. was examined by using Tween 20. As shown In addition to 5ƒÊmoles of MgSO4, l0ƒÊmoles of in Table IX, addition of 0.01ml. of 10% the indicated metal solutions were added. Tween 20 (total volume, 0.6ml.) was observed to stimulate the enzymatic synthesis of phy toene from [2-C14] mevalonic acid.

DISCUSSION The cell-free extract of a mutant of Staphylococcusaureus, which has been proved to accumulate phytoene instead of bright-colored carotenoids, was confirmed to be capable of synthesizing this colorless carotenoid from mevalonic acid. The effect of various sub stances added to this enzyme system was in vestigated. Among them, the stimulatory effect of addition of fluoride and nicotinamide should be attributed to the prevention of the breakdown of the presumed phosphorylated intermediates and cofactors (15). ATP was proved to be one of the essential

TABLE IX cofactors. From the results of preliminary Effect of Cysteine and Tween 20 upon the Biosynthesis experiments mevalonic acid was presumed to of Phytoene with Ammonium Sulfate-precipitated be phosphorylated first before conversion to Enzymes polymerized intermediates. ATP should be MnSO4, 10ƒÊmoles and 0.3ml. of 0.1M Tris required for the activation of mevalonic acid. buffer solution of enzymes precipitated with 90% Attempts are now being made to isolate and saturated ammonium sulfate solution (protein con identify phosphorylated intermediates. centration, 30.0mg./ml.) were used. Other TPNH was found to be an essential ingredients and conditions were the same as those cofactor in these systems. Lynen also found given in Table VIII. participation of TPNH in the biosynthesis of terpenoids and postulated the role of TPNH and a hydroquinone (16). By Popjak's as sumption, the presumed first synthesized C30- compound, dehydrosqualene, would be pro duced by polymerization of two molecules of pyrophosphorylated C15-units and the C30- compound thus formed would be hydrogenated to squalene with the participation of TPNH (17). The requirement of FAD in phytoene synthesis has not been previously reported and Enzymatic Synthesis of Bacterial Phytoene 251

its role is as yet uncertain. The most probable precipitated by 90% saturated ammonium role of FAD would be that it serves as an sulfate solution. essential constituent of related enzymes in this 3. In this biosynthesis, optimum pH was system. And it may act as a coenzyme when determined as 8.0 with Tris-HCl buffer and the saturated Coo-compound (lycopersene) is optimum concentration of ATP as l0ƒÊmoles dehydrogenated to phytoene. per 0.6ml. The stimulating effect of cysteine when 4. Addition of fluoride (10ƒÊmoles per incubation was carried out aerobically could 0.6ml.), nicotinamide (50ƒÊmoles per 0.6ml.), be explained as the result of its preventing and Tween 20 (final concentration; 0.17%) the oxidation of SH enzyme(s). The slight was shown to be stimulatory to the biosyn inhibition by cysteine in a nitrogen atmosphere thesis of phytoene. can be attributed to its combination with the 5. Among metal cations, Mn++ was substrates in competition with SH enzyme(s) practically the only one which stimulated the in this system. biosynthesis of phytoene by these systems. Mn++ and Mg++ were also proved to be 6. Anaerobic conditions were found to stimulatory in the biosynthesis of phytoene by favor the formation of phytoene. Cysteine these systems. The optimum pH was found also stimulates the biosynthesis of phytoene to be 8.0 for the biosynthesis of phytoene from only when incubation was carried out mevalonic acid. The fact that phosphate aerobically. buffer of the same pH suppresses the formation of phytoene can be ascribed to the precipita The author wishes to thank Prof. S. Tanaka for tion of Mg++ and Mn++ with phosphate ion his valuable advice and Prof. O. Hayaishi for the use even in the presence of ATP. of equipment throughout this study. Throughout this investigation, the total radioactivity of unsaponifiable substance was REFERENCES observed to be much higher than that of (1) Suzue, G., and Tanaka, S., Science, 129, 1359 phytoene. This may be ascribed to the pre (1959) sence of large amounts of the intermediate (2) Suzue, G., J. Biochem., 46, 1497 (1959)

C10-, C15-, C20-, and C40-compounds. (3) Suzue, G., Arch. Biochem. Biophys., 88, 180 (1960) Bacterial phytoene was shown not to form (4) Suzue, G., Biochim. et Biophys. Acta, 45, 616 any adduct either with urea in hot solution (1960) or with thiourea in cold solution. Therefore, (5) Tavormina, P. A., Gibbs, M. H., and Huff, J. W., J. Am. Chem. Soc., 78, 4498 (1956) identification of the incorporated radioactivity (6) Braithwaite, G., and Goodwin, T. W., Biochem. with phytoene in the present work mainly J., 67 13 p (1957) depended on the fact that it is non-separable (7) Park, R. B., and Bonner, J., J. Biol. Chem., 233, from carrier bacterial phytoene by chromato 340 (1958)

graphic separation. Contamination with other (8) Gloor, U., and Wiss, O., Arch. Biochem. Biophys., presumptive intermediates can be neglected 83, 216 (1959) due to their greatly differing physical pro (9) Amdur, B. H., Rifling, H., and Bloch, K., J. Am. Chem. Soc., 79, 2646 (1957) perties. (10) Lynen, F., 'International Symposium on Enzyme SUMMARY Chemistry,' Tokyo and Kyoto, P. 57 (1957) 1. Phytoene was synthesized from [2-C14] (11) Popjak, G., Gosselin, L., Youhotsky-Gore, I., and Gould, R. G., Biochem. J., 69, 238 (1958) mevalonic acid with cell-free extracts of a (12) Lowry, O., Rosebrough, N.J., Farr, A. L., and mutant of Staphylococcus aureus which ac Randall, R.J., J. Biol. Chem., 193, 265 (1951)

cumulated the carotenoid phytoene. (13) Rabourn, W.J., Qaackenbush, F. W., and Por 2. ATP, TPNH, FAD, and Mn++ are ter, J. W., Arch. Biochem. Biophys., 48, 267 (1954)

essential cofactors for the biosynthesis of phy (14) Suzue, G., Biochim. et Biophys. Acta, 50, 593 toene from mevalonic acid with enzymes (1961) 252 G. SUZUE

(15) Kornberg, A., and Lindberg, 0., J. Biol. Chem., I., Angew. Chem., 70, 738 (1958) 176, 665 (1948) (17) Popjak, G., and Cornforth, J. W., Advances in (16) Lynen, F., Eggerer, H., Henning, U., and Kessel, Enzymol., 22, 281 (1960)