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.