- Email: [email protected]
Characterization of undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum
ARCHIVES
OF
HIOCHKMISTRY
Characterization
AND
161,
BIOPHYSICS
375-383
of Undecaprenyl
Pyrophosphate
Lacfobacillos JIICHAEL Utriuersity
V. KEENAN
of Florida,
(1974)
Synthetase
from
planfarum’ CHARLES
AND
College of Arts Gainesville, Received
M. ALLEN,
and Sciences, Departmellt Florida Sd610 August
JR.
of Biochemistry,
9, 1973
A soluble long-chain polyprenyl pyrophosphate synthetase has been isolatcdfrom Lactobacillus plantarum and partially purified by DEAE-cellulose chrombtography in 1% Triton X-100. This enzyme catalyzes the synthesis of polyprenyl pyrophosphate from farnesyl pyrophosphate andA%sopentenyl pyrophosphate. The enzyme displays a requirement for farnesyl pyrophosphate and Triton X-series detergents. Treatment of polyprenyl pyrophosphate with Css-isoprenyl pyrophosphate phosphatase (Micrococcus Zysodeikticus) yielded polyprenyl monophosphate. Subsequent treatment of this product with a crude phosphatase from baker’s yeast resulted in the formation of free polyprenol, which was characterized by thin layer chromatography and exhibited Rfs which corresponded to those of authentic undecaprenol isolated from Lactobacillzts plantarum. Reverse phase cochromatography of the enzymically produced polyprenol and authentic undecaprenol indicated that the major enzymic products were undecaprenol and probably a longer chain polyprenol.
A partially purified soluble enzyme preparation from ill. lysodeikticus which synthesizes C&- to Cdo-polyprenyl pyrophosphates (7), using A3-isopentenyl pyrophosphate and farnesyl pyrophosphate as substrates, has been investigated further (S), and evidence was obtained for the synthesis of an undecaprenyl monophosphate. A cell-free lysate from Lactobacillus plantarum (9) has been reported to synthesize both undecaprenol and hexaprenol from mevalonate. We would like to report here the partial purification from L. plantarum of an undecaprenyl pyrophosphate synthetase which apparently is a soluble enzyme.
The role of undecaprenyl pyrophosphates as carriers of sugars in the formation of bacterial cell wall polysaccharides has been well established (1). Recent reports indicate that the enzymes t’hat, metabolize the undecaprenol or its phosphate esters are membrane associated, e.g. C55-isoprenyl pyrophosphate phosphatase (2) from Micrococcus lysodeikticus, C&-isoprenoid alcohol phosphokinase (3) and C&isoprenoid alcohol phosphatase (4) from Staphylococcus aureus. I?I vivo labeling experiments with mevalonate have been reported (5) indicating that the biosynthesis of the Cb6-polyprenol, bact oprcnol , in Lactobacillus casei takes place in the mesosome and plasma membranes. Furthermore, a particulate fraction which synthesizes polyprenyl pyrophosphate, identical to antigen carrier lipid, using A3-isopentenyl pyrophosphate and farnesyl pyrophosphat’e, has been described for Salmonella newington (6). 1 This National
work was supported Science Foundation
by a grant (GB-34246).
from
EXPERIMENTAL
Materials. Freeze-dried M. lysodeikticus was obtained from the Miles Chemical Company; pancreatic deoxyribonuclease from Calbiochem; and lysoayme and Escherichia coli alkaline phosphatase from the Sigma Chemical Company. Farnesol was purchased from ICN Chemical Corporation,
the 375
Copyright 811 rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
PROCEDURES
KEENAN and @-methylallyl chloride was obtained from Eastman Organic Chemicals. Sorbents for chromatography were: Whatman DEAE-cellulose (DE-23), Kieselguhr G (E. Merck), and silica gel G without gypsum on plastic sheets from Brinkmann Instruments. Silica gel G precoated glass plates were obtained from Applied Science Lab. and Sephadex G-25 from Pharmacia. Methods. Thin layer chromatography was carried out on silica gel G plates or sheets using the following solvent systems: Solvent I, 2-propanol/ coned NHIOH/water (4:3:l,v/v); Solvent II, lpropanol/concd NHaOH/water (6:2:2,v/v); Solvent III, chloroform; Solvent IV, benzene/ethyl acetate (9: 1, v/v); Solvent V, diisobutylketone/ acetic acid/water (8:5:l,v/v); and Solvent VI, nheptane/ethyl acetate (S:l,v/v). Reverse phase thin layer chromatography was carried out on Kieselgubr G plates (prepared by immersing the plate in a 5% (v/v) Solution of paraffin oil in petroleum ether, b.p. 30-60”) in Solvent system VII acetone/Hz0 (23:2,v/v). Farnesol (approximately 64% trans,trans, and 36% cis,trans as determined by gas chromatography) was phosphorylated according to a general method described by Popjak et al. (10). Farnesyl pyrophosphate was isolated as described by Holloway and Popjak (11) or by crystallization from a solution of 0.01 M NH,OH in methanol. The product chromatographed with Rf 0.32 in Solvent I on silica gel plates. 3-Methyl-3-[l-‘%]butenoic acid was prepared by carbonation of the Grignard of /3-methylallyl chloride with i4CO2 in tetrahydrofuran, and the resulting acid was reduced to 3-methyl-3-[1-14C]butenol with LiAlH,, as descirbed by Yuan and Bloch (12). The alcohol was phosphorylated as described for farnesol. A3-[I-i%]Isopentenyl pyrophosphatea (sp act 0.45 ,.&i/pmole) was isolated by chromatography over DEAE-cellulose with a gradient of 0.01 M (NH,),CO, to 0.1 M (N&)&03 in methanol, as described by Dugan et al. (13). The radioactive product chromatographed with RI 0.47 in Solvent II on silica gel plates. Undecaprenol was isolated from I,. plantarum by a procedure similar to that described by Gough et al. (14). Twenty-eight grams of washed cells from a 36.hr culture were suspended in a solution containing 80 ml of 1% pyrogallol in methanol and 30 ml of 60% aqueous KOH and refluxed under nitrogen for 2 hr. The mixture was cooled in an ice bath, and 420 ml of 1.7 M NaCl was added. This mixture was extracted 3 times with 500 ml of ether/ 2 3.Methyl-3-[lJ%]butenyl pyrophosphate will be referred to as A3-[l-14C]isopentenyl pyrophosphate.
AND
ALLEN
petroleum ether, b.p. 30-60” (l:l,v/v). The combined ethereal solutions were washed with water to neutrality, dried over Na&OI, and evaporated to dryness with a rotary evaporator under vacuum. The fragrant yellow oil was then chromatographed by thin layer chromatography on silica gel G plates in Solvent III. A portion of the plate was trea.ted with anisaldehyde reagent (15) to visualize the polyprenols. A broad region of silica gel around Rf 0.66 was scraped from the plate and extracted with 150 ml of ethyl ether. This ether extract was concentrated and applied to a second silica gel G plate, and the plate was developed in Solvent IV. The major component in Solvent IV had an RI 0.74. This component was scraped from the plate and characterized as undecaprenol. It chromatographed as one component with Rfs 0.74 and 0.96 on silica gel G sheets in Solvents IV and V, respectively, and RI 0.55 on reverse phase chromatography in Solvent VII. Mass spectral analysis of this sample indicated it was a C55polyprenol which fragmented as previously described (14). The molecular ion appeared at m/e 766. Other major peaks were at m/e 748 (M-HzO)+ ion, 680, 611, 543, 475, 407, 339, 271, 203, 135, 69 (base peak). Enzyme preparations. Lactobacillus plantarum (ATCC 8014) was grown at 30°C in stationary culture using the semisynthetic medium described by Skeggs et al. (16). For the general preparation of enzyme a 12-hr culture was harvested and washed with 0.1 M Tris buffer (pH 7.5). A cell-free lysate was prepared according to the general procedure described by Habbal and Durr (17). Twenty grams of washed cells were suspended in 375 ml of 1 M sucrose containing 0.03 M Tris buffer (pH 7.9), 1 X low3 M glutathione, and 90 mg of lysozyme. The suspension was incubated at 37°C for 45 min with frequent stirring. Subsequent steps for the isolation of this enzyme and other enzyme activities were carried out at 0-4°C except as noted. The cells were then centrifuged at 48,OOOg for 15 min, and the resulting pellet was shocked by suspension in 75 ml of ice-cold 0.01 M Tris-maleate buffer (pH 6.0). This suspension was then treated with deoxyribonuclease at 25°C as previously described and centrifuged at 20,OOOg for 30 min. The resulting supernatant was further centrifuged at 48,OOOg for 30 min. This supernatant, or one prepared by centrifugation at 100,OOOg for 1 hr, was used for DEAE-cellulose chromatography. More activity can be washed from the 20,OOOg pellet by resuspending it in 50 ml of 0.01 M Tris buffer (pH 7.0) and centrifuging at 48,OOOg for 30 min. Some activity, however, always remains in the pellet. This activity can be solubilized by suspension of the pellet in l-2 vol of 0.01 M Tris buffer (pH 7.5) con-
UNDECAPRENYL
PYROPHOSPHATE
FRLCTION
SYNTHETASE
377
NUMBER
1. Chromatography of 48,000g supernatant on DEAE-cellulose in Triton X100. Methods of chromatography and enzyme assay are described in the experimental section. Aliquots of 0.2 ml each were taken for determination of protein concentration and enzyme activity from the 14-ml fractions. The trichloroacetic acid hydrolysis in the assay procedure was carried out at 60°C for 10 min. Closed circles represent A660 nm (protein), and open circles represent cpm observed in the enzymic assay. FIG.
taining 0.01 M MgC12, lyO Triton X-100, and 0.01 M mercaptoethanol. The 48,000g supernatant, containing 405 mg of protein, was applied to a 40 X 2.5 cm DEAE-cellulose column equilibrated with 0.01 M Tris buffer (pH 7.0) containing lro Triton X-100. The protein was eluted from the column with a linear gradient of NaCl (CO.6 M) in the Tris-Triton X-100 buffer, using 750 ml of elutant (Fig. 1). Protein was determined by the method of Lowry el a.l. (18). A precipitate formed during the protein determination due to the presence of Triton X-100 and was removed by centrifugation before determining the optical density at 660 nm. The enzyme activity was measured by the general procedure indicated below. Those fractions containing activity were pooled, concentrated threefold by ultrafiltration, and stored at -20°C. The membrane preparation used as a source of C:s-isoprenyl pyrophosphate phosphatase (2) was obtained from freeze dried !W. Zysodeidficus. Ten grams of cells were treated with lysozyme and deoxyribonuclease according to a previously described procedure (7). The crude broken cell suspension was centrifuged at 27,OOOg for 20 min. The resulting pellets were resuspended in 0.05 M Tris buffer (pH 7.5) and sonicated with a Biosonik II for 5 min while cooled in an ice-salt water slurry. The sonicate was centrifuged at 27,000g for 20 min. The supernatant was t.hen centrifuged at lOO,OOOq
for 1 hr. The resulting pellets were resuspended in 0.05 M Tris buffer (pH 7.5) containing0.1 I/I EDTA, I mM ascorbic acid, and centrifuged at 100,OOOg for 1 hr. This membrane pellet was resuspended in 0.05 M Tris buffer (pH 7.5) containing 1 mM ascorbic acid and used as the source of the Cis-isoprenyl pyrophosphate phosphatase. A second phosphatase activity was prepared from baker’s yeast (15 g) which had been SLW pended at room temperature in 250 ml of lO”x sucrose for 1.5 hr. The yeast were harvested by centrifugation and mixed with approximately 15 g of washed sea sand. Sufficient 0.05 M Tris buffer (pH 7.5) was added to bring the total volume to 40 ml. This mixture was ground at 0°C for 10 min and then centrifuged at 8OOg for 10 min. The supernatant was used as the crude phosphatase preparation from yeast (8). Enzyme assay. The assay solution contained in a final volume of 0.5 ml: 0.21 mM Tris buffer (pH 7.0), 50 pM farnesyl pyrophosphate, 30 SM A3-[lWlisopentenyl pyrophosphate (30,000 dpm), 0.5%. Triton X-100, enzyme, and a variable amount of MgCla depending on the experiment. The reaction mixtures were incubated for 30 min at 30°C. unless otherwise indicated. The reaction products were acid hydrolyzed with 25% trichloroacetic acid at 100°C for 30 min, neutralized with base, and extracted with pet’roleum ether as previously described (7) unless otherwise indicated. A 5-ml
378
KEENAN
AND ALLEN
aliquot of the petroleum ether extract was evaporated to dryness and assayed for radioactivity in toluene-Omnifluor (New England Nuclear) scintillation fluid. Product isotation. The following represents a typical incubation for the formation of radioactive product to be used in the purification and characterization of polyprenyl pyrophosphate. A reaccontaining A3-[1J4C]isopentenyl tion mixture pyrophosphate (187,500 dpm), 0.05 mM farnesyl pyrophosphate, 0.5?& Triton X-100, 0.01 M MgClz, 0.1 M Tris buffer (pH 7.0), and 0.69 mg of DEAEpurified enzyme in a total volume of 3.0 ml was incubated at 37°C for 5 hr. The entire reaction mixture was chromatographed in 0.2 M Tris buffer (pH 7.5) on a Sephadex G-25 column (VO = 14 ml), collecting 5-drop fractions. Aliquots from the column were assayed for radioactivity in a dioxanebased scintillation fluid (19). The polyprenyl pyrophosphate was isolated from the protein-product mixture obtained by Sephadex G-25 chromatography (Fig. 2) by extraction with I-butanol or CHCla-methanol (2: 1, v/v). The solvents were removed from the product by rotary evaporation under vacuum at room temperature to avoid degradation of the product to less polar uncharacterized products. The thin layer radiochromatograms illustrated in Figs. 4,5, and 6 were obtained using a Packard
Model ing at tector on an
7201 Radiochromatogram Scanner recorda chart rate of 0.5 cm/min and full scale deresponse of 300. Mass spectra were obtained AEI-MS30 mass spectrometer. RESULTS
Enzyme Purifccation The profile for elution of protein and enzyme from DEAE-cellulose illustrated in Fig. 1 is essentially the same for a 48,000g supernatant, 100,OOOg supernatant, or a detergent extract of the 20,OOOgpellet. The peak of enzyme activity routinely corresponded to a molarity of 0.35-0.45 M in NaCl in the eluting solvent. DEAE-cellulose chromatography resulted in approximately a threefold purification of the enzyme. If the DEAE-cellulose chromatography was carried out in the absence of Triton X-100, the eluted protein had an enzyme activity less than one qua.rter of that observed when detergent. was included in the eluting solvent. Before evaluation of the enzyme activity, detergent was always added to the enzyme assay mixtures. Attempted purification of the enzyme on hydroxy-
6
40
80 FRACTION
120
160
NUMBER
FIG. 2, Sephadex G-25 chromatography of reaction mixtures after incubation of enzyme with A*-[l-14C]isopenteny1 pyrophosphate with and without farnesyl pyrophosphate. Conditions for this incubation mixture are described in the experimental section. The closed circles represent the observed radioactivity in the presence of 0.05 mM farnesyl pyrophosphate, and the open circles represent radioactivity observed when farnesyl pyrophosphate is omitted from the reaction mixture.
UNDECAPRENYL
PYROPHOSPHATE
apatite, by gradient elution in 0.014.10 M potassium phosphate buffer (pH 7.5) in the presence of 1% Triton X-100, resulted in a marked loss in enzyme activity. Phosphate, however, does not inhibit t’he enzyme activity. Reaction Requirements Incubation of farnesyl pyrophosphate and A3-[lJ4C]isopentenyl pyrophosphat’e with the enzyme isolated from DEAE-cellulose chromatography resulted in the formation of acid labile polyprenyl pyrophosphate. Table I illustrates that product formation was dependent on the presence of farnesyl pyrophosphate and Triton X-100. It is also apparent that the enzyme is only partially inactivated by the ethanol extraction procedure used to remove detergent from the enzyme. Preliminary evidence indicates an apparent divalent cation requirement since the enzyme is completely inhibited by 10, 1, and TABLE RUCTION
Reaction
I
REQUIREMENTS FOR THE SYNTHESIS POLYPRENYL PYROPHOSPHATE~ components
Complete Minus farnesyl phosphate Minus Triton Minus MgC12 Boiled enzyme
pyroX-100
Enzyme
OF
fraction
DEAEcellulose fraction #pm)
Ethanol washed DEAEcellulose fraction (dpm)
13,480 209
4,220 60
13,530 63
77 6,120 45
5 The complete reaction vessel contained the same concentration of components as indicated in the general assay procedure with the addition of M&l* (10 mM) and 0.34 mg of protein except as indicated. Each reaction mixture was incubated at 30°C for 30 min and analyzed as described in the general assay procedure. The ethanol washed DEAE-enzyme was prepared by treatment of the DEAE enzyme with 2 vol of ice-cold ethanol, washing the resulting precipitate twice with 2 vol of ethanol, and resuspending enzyme in 1 vol of 0.01 M Tris buffer (pH 7.0). This procedure removes the Triton X-100 from DEAE-enzyme.
379
SYNTHETASE TABLE
II
ACTIVATION OF ETHANOL WASHED SYNTHBTASE TRITON X-SERIES DETERGENTS~ Detergent Triton Triton Triton Triton None
BY
Radioactivity in product (dpm)
X-45 X-100 X-102 X-114
201 477 478 635 21
0 Each reaction vessel contained the same concentration of components as indicated in the general assay procedure (without magnesium) using 0.12 mg of enzyme and the detergents at a final concentration of 1%. Each reaction mixture was incubated at 30°C for 30 min and analyzed as described in the general assay procedure. The ethanol washed enzyme was prepared as described in Table I.
0.1 mu EDTA in the absence of added magnesium. The inhibition observed at 10 mM EDTA can be overcome by the addition of 20 rnM MgCL. Table II illustrates that several detergents of the Triton X-series will activate the ethanol-washed DEAE-enzyme. Under the same assay conditions, a variety of other detergents, Brij 35 (1.7 %), Brij 58 (1.7 %), Tween 80 (l%), deoxycholate (l%), and sodium dodecyl sulfate (l%), were all ineffective in stimulating the ethanol washed enzyme. Product Isolation The polyprenyl pyrophosphate can be separated from radioactive starting material, A3-[1-14C]isopentenyl pyrophosphate, by chromatography on Sephadex G-25 (Fig. 2). The polyprenyl pyrophosphate cochromatographs with protein emerging in the void volume. Proof that the radioactivity associated with the protein is product specific and not due to nonspecific binding of A3[lJ4C]isopentenyl pyrophosphate is illustrated by the small amount of radioactivity associated with the protein when farnesyl pyrophosphate is omitted from the reaction vessel. Furthermore, the radioactive product associated with the protein can be removed by extractionwitk e&her l-bt&rtol or chloroform-methanol (2: 1, v/v). A eom-
380
KEENAN
AND
parison of the chromatographic properties on thin layer chromatography (Solvent V) of this product (R, 0.47) with A3-[l-‘*Clisopentenyl pyrophosphate (Rf 0.01) indicated that there is no isopcntenyl pyrophosphatc bound to protein. Product
Characterization
The rate of hydrolysis of the polyprenyl pyrophosphate, at 100°C in 17 and 25% trichloroacetic acid, was determined by measuring the formation of petroleum ether soluble radioactivity (Fig. 3). The maximum petroleum ether soluble radioactivity was formed after about 20 min at each concentration of acid and represents about 92 % (25% trichloroacetic acid) of the total radioactivity associated with the product. The radioactive product resulting from this acid hydrolysis chromatographs as essentially one component by thin layer chromatography on silica gel G sheets in solvents IV, V, and VI with Rfs of 0.92, 0.96, and 0.93, respectively. Similarly, acid hydrolysis of the poly-
ALLEN
prenyl pyrophosphate in 25 % trichloroacetate acid at reduced temperature (6O’C) for 10 min renders greater than 90% of the radioactivity as petroleum ether soluble material. The products of this hydrolysis chromatograph on silica gel G sheets as two major components with Rfs 0.92 and 0.83 in Solvent IV. The faster and slower moving components are probably an undecaprenyl hydrocarbon and tertiary alcohol, respectively, as evidenced by similar chromatographic properties observed by Stone and Strominger (20) for the acid hydrolysis products of undecaprenyl pyrophosphate. The polyprenyl pyrophosphate is completely resistant to alkaline hydrolysis in 10% KOH for 1 hr at 100°C. Polyprenyl pyrophosphates synthesized by enzymes from M. lysodeilcticus are resistant to E. coli alkaline phosphatase at 37°C (7, 8). The polyprenyl pyrophosphate isolated here is completely resistant to the action of E. coli alkaline phosphatase at pH 9.2 in the presence or absence of Triton X-100 at 37” and 50°C. The alkaline phos-
6
MINUTES
FIG. 3. The acid lability of the enzymic product at 100°C. The polyprenyl pyrophosphate was prepared by the procedure described in Fig. 2. The protein-product mixture excluded from the Sephadex G-25 was divided into several 0.6-ml aliquots. To each aliquot was added either 0.3 ml or 0.6 ml of 500/e trichloroacetic acid. All the acidified aliquots were then heated in a boiling water bath, and at various periods of time samples were removed, cooled in an ice bath, neutralized with 0.3 ml or 0.6 ml of 5 M NaOH, respectively, and extracted with petroleum ether as in the general enzyme assay. The closed and open circles represent product formation in the presence of 17% and 257, trichloroacetic acid, respectively.
UNDECAPRENYL
PYROPHOSPHATE
SYNTHETASE
381
for the polyprenyl monophosphate (Fig. 4) (20). Cochromatography of the product of the i?f. lysodeilhus membrane treated polyprenyl pyrophosphate with untreated polyprcnyl pyrophosphate is illustrat’ed in Fig. 5. When the polyprenyl monophosphatc was treated with a crude yeast’ homogenate a radioactive component was obt’ained which chromatographed in Solvents IV and V with R/s 0.74 and 0.94, respectively, that correspond to those of undecaprenol isolated and FRONT ORl61N characterized from L. plantarum (Kg. 6AFIG. 4. Chromatogram of polyprenyl monoB).3b All of the observed radioactivity on phosphate obtained by C&s-isoprenyl pyrophoschromatogram A, Fig. 6, was cluted from phate phosphatase treatment of L. plantarum the silica gel, mixed with authentic unpolyprenyl pyrophosphate. The polyprenyl pyrophosphate isolated as described in the experidecaprenol, and subjected to reverse phase mental section was incubated for 5 hr, at 37°C in chromatography in Solvent VII (Fig. 7). 0.6 ml of a solution containing 0.1 ml of M. ZysoA component was observed which codeikticus membrane preparation (1.92 mg), Tris chromatographed with authentic undecbuffer (pH 7.5), 95 rmoles; EDTA, 7 pmoles; aprcnol. The lack of coincidence between Triton X-100, 1%; MgC&, 70 rmoles. The product authentic undecaprenol and all of the radiowas isolated from the reaction mixture by extracact’ivity associated with the enzymatic tion twice with an equal volume of I-butanol. The polyprenol product indicates that there is butanol extract was washed with 0.5 vol of water more than one component present with an and concentrated. The product (approximately 10,000 dpm) was dissolved in ethanol and spotted Rf similar to that of undecaprenol. During on a silica gel G sheet,. The sheet was developed in t’he large scaleisolation of undecaprenol from Solvent V. L. plantarum, we have never detected more than one component (undecaprenol) having phatase is active on the artificial substrate this Rf. It is possible that’ isomeric or longer p-nitrophenyl phosphate under the same conditions. The butanol or chloroform-methanol extracted product has been further charactcrized dures.
by additional chromatographic The product chromatographs
procein
Solvent V with Rf 0.47, J+hich is what is expected for a polyprenyl pyrophosphate3” (20). Treatment of this polyprenyl pyrophosphate with M. lysodeilcticus C&-isoprenyl pyrophosphate phosphatase (2) results in the formation of a radioactive component with an R, 0.60 in Solvent V corresponding to what would be expected 3 (a) Studies by Kenneth Meyer (unpublished work) in this laboratory indicate that the C3&& polyprenyl pyrophosphates synthesized by the polyprenyl pyrophosphate synthetase from M. Zysodeikticus have RJS from 0.2 to 0.34 in the same chromatographic system. (b) Treatment of the pyrophosphates from M. Gas-Go polyprenyl lysocleikticus with the yeast homogenate gave a polyprenol product which chromatographed in solvent IV with an RI of 0.47.
FRONT
ORl6lN
5. Chromatogram of a mixture of L. plantarum polyprenyl pyrophosphate and polyprenyl monophosphate. Polyprenyl monophosphate was prepared from polyprenyl pyrophosphate as described in the legend of Fig. 4. Approximately 8,000 dpm each of polyprenyl monophosphate and polyprenyl pyrophosphate were combined and co-chromatographed on a silica gel G sheet in Solvent V. FIG.
382
KEENAN
AND
II.4 0 0, FRONT
ORIGIN
6. Chromatograms of the products formed by sequential action of Css-isoprenyl pyrophosphate phosphatase and yeast phosphatase on L. FIG.
plantarum polyprenyl pyrophosphate. Polyprenyl pyrophosphate was incubated with membranes from M. lysodeikticus (0.98 mg) as described in Fig. 4. After 5 hr, 120 pmoles MgClz and 1 ml of the yeast preparation were added, and the mixture was incubated an additional 5 hr at 37°C. The product was extracted from the reaction mixture
ALLEN
hydrolyzed by M. lysodeikticus membranes to polyprenyl monophosphate in a manner characteristic of the action of Ce5-isoprenyl pyrophosphate phosphatase on undecaprenyl pyrophosphate (20). Finally, the hydrolytic product resulting from the treatment of polyprenyl monophosphate with yeast homogenate chromatographs primarily as undecaprenol (Figs. 6 and 7). Its chromatographic mobility is similar to that observed for a C&,-polyprenol isolated in a similar manner from a mixture of polyprenyl pyrophosphates produced by the M. lysodeikticus synthetases (8). Although several of the solvent systems used are not highly selective with regard to distinguishing chain length, the chromatographic mobilities of the radioactive alcohol on reverse phase chromatography illustrates cochromatography of the product with undecaprenol. Although the major compo-
with three 5-ml portions of CHCY-methanol, 2:l (v/v), and the extract was washed with 1 ml of water.
The
product
(approximately
5,000
dpm)
was divided into two equal portions and chromatographed on a silica gel G sheet in Solvent (Chromatogram A) and Solvent V (Chromatogram
IV
B).
chain polyprenols are formed that either are not found as free polyprenols following base hydrolysis of L. plantarum or are produced under the unregulated conditions of our in vitro experiments. DISCUSSION We have presented evidence for the isolation of a polyprenyl pyrophosphate synthetase from L. plantarum. The primary enzymic product has been characterized as
undecaprenyl pyrophosphate. The polyprenyl pyrophosphate chromatographs similarly to undecaprenyl pyrophosphate isolated from AI. lysodeikticus (20). The conditions for its acid hydrolysis and t,he chromatograptiic properties of the resulting hydrolysis products are characteristic of undecaprenyl pyrophosphates.
The extracted polyprenyl product has been further characterized by the chromatographic properties of its enzymic hydrolysis products. Polyprenyl pyrophosphate was
9 ORIGIN
FRONT
FIG. 7. Reversephasechromatogramof polyprenol formed by sequential treatment of polyprenyl pyrophosphate with Csh-isoprenyl pyrophosphate phosphatase and yeast phosphatase. The polyprenol (approximately 1,000 dpm) was eluted from the chromatogram illustrated in Fig. 6A and subjected to analytical scale reverse phase thin layer chromatography. Some variation in the RI of a sample has been experienced with reverse phase thin layer chromatography on this scale. Accordingly, authentic undecaprenol (L. plantarum) was added to the enzymatic product (A) prior to chromatography. A second undecaprenol sample (B) was also chromatographed. The plate was visualized with 12, and the labeled portion of the plate was cut into 0.6 X 2 cm bands and counted in toluene scintillation fluid. The unlabeled sample of undecaprenol was visualized with anisaldehyde reagent to confirm the results obtained with 1~.
UNDECAPRENYL
PYROPHOSPHATE
ncnt is most likely undecaprenol, the prescnce of related compounds such as isomeric or longer chain polyprenols is possible. It should be pointed out, however, that in vivo (14) and in vitro (9) stud& on the biosynthesis of polyprenols indicate that undecaprenol is the major and highest molecular weight polyprenol synthesized by L. plantarum.
Scphadcx G-25 chromatography of the complete reaction mixture permits a rapid and complete separation of the enzymic product from starting materials. Although the product accompanies the protein in the void volume of the column, no definitive evidence has been presented t,o indicate if any interaction between protein and product exists. Previous work of Kurokawa et al. (8) indicates that long chain polyprenyl pyrophosphates are excluded from Sephadex G-25 in the absenceof protein. Soncovalent associat’ion of polyprenyl pyrophosphat’es with protein has previously been described by Allen et al. (7) for the C&C40 polyprcnyl pyrophosphate synthet’ase from M. lysodeikticus, Oster and West (21) and Ogura et al. (22) for gcranylgeranyl pyrophosphate synthetase, and Rilling (23) for presqualene pgrophosphate synthetase. The enzyme, like other polyprenyl pyrophosphate synthetases, demonstrates a requirement for farnesyl pyrophosphatr and divalent cation. In addition, enzyme activ y is also dependent on the addition of detergent. The nonionic detergents of the Triton X-series are effective, whereas, other nonionic and ionic detergents tested do not’ activate the enzJTme.This requirement for the presence of detergent’ is unique among t,he polyprenyl pyrophosphate synthetases thus far described, since neither the C&Z4,, polyprenyl pyrophosphatc synthetase (7, 8) nor the undecaprenyl pyrophosphate synthcttases (6, 8, 9) require the presence of detergent for cnzymic activit’y. Studies are continuing t’o further charact’erize both the undccaprcnyl pyrophosphate and its synthctase. ACKNOWLEDGMENTS We are grateful to Dr. Roy out the mass spectral analyses nol.
King for carrying of the undecapre-
SYNTHETASE
383
REFERENCES 1. ROTHFIFLD, L., AND ROMEO, 1). (1971) Bacteriol. Rev. 36, 14-38. 2. GOLDMAN, R., BND STROMINGER, J. L. (1972) J. Biol. Chem. 247, 5116-5122. 3. HIGASHI, Y., SIEWERT, G., AND STROMINGER, J. L. (1970) J. Biol. Chena. 246, 3683-3690. 4. WILLOUGHBY, E., HIGASHI, Y., AND STROMINGER, J. L. (1972) J. Biol. Chem. 247, 51135115. 5. THORNE, K. J. I., AND BARKER, D. C. (1971) Biochem. J. 122, 45P46P. 6. CHRISTENSON, J. G., GROSS, S. K., AND ROBBINS, P. W. (1969) J. Biol. Chem. 244, 54365439. 7. ALLEN, C. M., AL~ORTH, W., MACCR~E, A., AND BLOCH, K. (1967) J. Biol. Chem. 242, 1895-1902. 8. KUROK~TVA, T., OGURA, K., AND &TO, S. (1971) Biochem. Biophys. Res. Commun. 46, 251-257. 9. DURR, I. F., END HABBAL, M. Z. (1972) Biothem. J. 127, 345-349. 10. POPJAK, G., CORNFORTH, J. W., CORNFORTH, H. H., R~H~GE, R., AND GOODMSN, D. S. (1962) J. Biol. Chem. 237, 56-61. 11. HOLLOWBY, P. W., AND POPJaK, G. (1967) Biochem. J. 104, 57-70. 12. YUAN, C., AND BLOCH, K. (1959) J. Biol. Chem. 234, 2605-2608. 13. DUGAN, R. E., Rasson-, E., AND PORTER, J. W. (1968) Anal. Biochem. 22,249-259. 14. GOUGH, D. P., KIRBY, A. L., RICHARDS, J. B., AND HEMMING, F. W. (1970) Biochem. J. 118, 167-170. 15. MCSTVEENEY, G. P. (1965) J. Chromatogr. 17, 183-185. 16. &EGGS, H. R., WRIGHT, L. D., CRESSON, E. L., &1acRa~, G. D. E., HOFFMAN, C. H., WOLF, D. E., .LND FOLI~ERS, K. (1956) J. Bacterial. 72, 519-524. 17. HA~EL~L, Z., AND DURR, I. F. (1969) J. Bacterial. 100, 1409-1410. 18. Lowax-, 0. H., ROSERROUGH, N. J., F.~RR, 8. L., AND RANDALL, It. R. (1951) J. Biol. Chem. 193, 265-275. 19. ALLEN, C. M. (1972) Biochemistry 11, 21542160. 20. STONE, K. J., AND STROMINGER, J. L. (1972) J. Biol. Chem. 247, 5107-5112. 21. OSTER, M. O., AND WEST, C. A. (1968) Arch. Biochem. Biophys. 12’7, 112-123. 22. OGURA, K., SHINKA, T., AND SETO, S. (1972) J. Biochem. 72, 1101-1108. 23. RILLING, 1%. C. (1970) J. Lipid Res. 11, 480485.
OF
HIOCHKMISTRY
Characterization
AND
161,
BIOPHYSICS
375-383
of Undecaprenyl
Pyrophosphate
Lacfobacillos JIICHAEL Utriuersity
V. KEENAN
of Florida,
(1974)
Synthetase
from
planfarum’ CHARLES
AND
College of Arts Gainesville, Received
M. ALLEN,
and Sciences, Departmellt Florida Sd610 August
JR.
of Biochemistry,
9, 1973
A soluble long-chain polyprenyl pyrophosphate synthetase has been isolatcdfrom Lactobacillus plantarum and partially purified by DEAE-cellulose chrombtography in 1% Triton X-100. This enzyme catalyzes the synthesis of polyprenyl pyrophosphate from farnesyl pyrophosphate andA%sopentenyl pyrophosphate. The enzyme displays a requirement for farnesyl pyrophosphate and Triton X-series detergents. Treatment of polyprenyl pyrophosphate with Css-isoprenyl pyrophosphate phosphatase (Micrococcus Zysodeikticus) yielded polyprenyl monophosphate. Subsequent treatment of this product with a crude phosphatase from baker’s yeast resulted in the formation of free polyprenol, which was characterized by thin layer chromatography and exhibited Rfs which corresponded to those of authentic undecaprenol isolated from Lactobacillzts plantarum. Reverse phase cochromatography of the enzymically produced polyprenol and authentic undecaprenol indicated that the major enzymic products were undecaprenol and probably a longer chain polyprenol.
A partially purified soluble enzyme preparation from ill. lysodeikticus which synthesizes C&- to Cdo-polyprenyl pyrophosphates (7), using A3-isopentenyl pyrophosphate and farnesyl pyrophosphate as substrates, has been investigated further (S), and evidence was obtained for the synthesis of an undecaprenyl monophosphate. A cell-free lysate from Lactobacillus plantarum (9) has been reported to synthesize both undecaprenol and hexaprenol from mevalonate. We would like to report here the partial purification from L. plantarum of an undecaprenyl pyrophosphate synthetase which apparently is a soluble enzyme.
The role of undecaprenyl pyrophosphates as carriers of sugars in the formation of bacterial cell wall polysaccharides has been well established (1). Recent reports indicate that the enzymes t’hat, metabolize the undecaprenol or its phosphate esters are membrane associated, e.g. C55-isoprenyl pyrophosphate phosphatase (2) from Micrococcus lysodeikticus, C&-isoprenoid alcohol phosphokinase (3) and C&isoprenoid alcohol phosphatase (4) from Staphylococcus aureus. I?I vivo labeling experiments with mevalonate have been reported (5) indicating that the biosynthesis of the Cb6-polyprenol, bact oprcnol , in Lactobacillus casei takes place in the mesosome and plasma membranes. Furthermore, a particulate fraction which synthesizes polyprenyl pyrophosphate, identical to antigen carrier lipid, using A3-isopentenyl pyrophosphate and farnesyl pyrophosphat’e, has been described for Salmonella newington (6). 1 This National
work was supported Science Foundation
by a grant (GB-34246).
from
EXPERIMENTAL
Materials. Freeze-dried M. lysodeikticus was obtained from the Miles Chemical Company; pancreatic deoxyribonuclease from Calbiochem; and lysoayme and Escherichia coli alkaline phosphatase from the Sigma Chemical Company. Farnesol was purchased from ICN Chemical Corporation,
the 375
Copyright 811 rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
PROCEDURES
KEENAN and @-methylallyl chloride was obtained from Eastman Organic Chemicals. Sorbents for chromatography were: Whatman DEAE-cellulose (DE-23), Kieselguhr G (E. Merck), and silica gel G without gypsum on plastic sheets from Brinkmann Instruments. Silica gel G precoated glass plates were obtained from Applied Science Lab. and Sephadex G-25 from Pharmacia. Methods. Thin layer chromatography was carried out on silica gel G plates or sheets using the following solvent systems: Solvent I, 2-propanol/ coned NHIOH/water (4:3:l,v/v); Solvent II, lpropanol/concd NHaOH/water (6:2:2,v/v); Solvent III, chloroform; Solvent IV, benzene/ethyl acetate (9: 1, v/v); Solvent V, diisobutylketone/ acetic acid/water (8:5:l,v/v); and Solvent VI, nheptane/ethyl acetate (S:l,v/v). Reverse phase thin layer chromatography was carried out on Kieselgubr G plates (prepared by immersing the plate in a 5% (v/v) Solution of paraffin oil in petroleum ether, b.p. 30-60”) in Solvent system VII acetone/Hz0 (23:2,v/v). Farnesol (approximately 64% trans,trans, and 36% cis,trans as determined by gas chromatography) was phosphorylated according to a general method described by Popjak et al. (10). Farnesyl pyrophosphate was isolated as described by Holloway and Popjak (11) or by crystallization from a solution of 0.01 M NH,OH in methanol. The product chromatographed with Rf 0.32 in Solvent I on silica gel plates. 3-Methyl-3-[l-‘%]butenoic acid was prepared by carbonation of the Grignard of /3-methylallyl chloride with i4CO2 in tetrahydrofuran, and the resulting acid was reduced to 3-methyl-3-[1-14C]butenol with LiAlH,, as descirbed by Yuan and Bloch (12). The alcohol was phosphorylated as described for farnesol. A3-[I-i%]Isopentenyl pyrophosphatea (sp act 0.45 ,.&i/pmole) was isolated by chromatography over DEAE-cellulose with a gradient of 0.01 M (NH,),CO, to 0.1 M (N&)&03 in methanol, as described by Dugan et al. (13). The radioactive product chromatographed with RI 0.47 in Solvent II on silica gel plates. Undecaprenol was isolated from I,. plantarum by a procedure similar to that described by Gough et al. (14). Twenty-eight grams of washed cells from a 36.hr culture were suspended in a solution containing 80 ml of 1% pyrogallol in methanol and 30 ml of 60% aqueous KOH and refluxed under nitrogen for 2 hr. The mixture was cooled in an ice bath, and 420 ml of 1.7 M NaCl was added. This mixture was extracted 3 times with 500 ml of ether/ 2 3.Methyl-3-[lJ%]butenyl pyrophosphate will be referred to as A3-[l-14C]isopentenyl pyrophosphate.
AND
ALLEN
petroleum ether, b.p. 30-60” (l:l,v/v). The combined ethereal solutions were washed with water to neutrality, dried over Na&OI, and evaporated to dryness with a rotary evaporator under vacuum. The fragrant yellow oil was then chromatographed by thin layer chromatography on silica gel G plates in Solvent III. A portion of the plate was trea.ted with anisaldehyde reagent (15) to visualize the polyprenols. A broad region of silica gel around Rf 0.66 was scraped from the plate and extracted with 150 ml of ethyl ether. This ether extract was concentrated and applied to a second silica gel G plate, and the plate was developed in Solvent IV. The major component in Solvent IV had an RI 0.74. This component was scraped from the plate and characterized as undecaprenol. It chromatographed as one component with Rfs 0.74 and 0.96 on silica gel G sheets in Solvents IV and V, respectively, and RI 0.55 on reverse phase chromatography in Solvent VII. Mass spectral analysis of this sample indicated it was a C55polyprenol which fragmented as previously described (14). The molecular ion appeared at m/e 766. Other major peaks were at m/e 748 (M-HzO)+ ion, 680, 611, 543, 475, 407, 339, 271, 203, 135, 69 (base peak). Enzyme preparations. Lactobacillus plantarum (ATCC 8014) was grown at 30°C in stationary culture using the semisynthetic medium described by Skeggs et al. (16). For the general preparation of enzyme a 12-hr culture was harvested and washed with 0.1 M Tris buffer (pH 7.5). A cell-free lysate was prepared according to the general procedure described by Habbal and Durr (17). Twenty grams of washed cells were suspended in 375 ml of 1 M sucrose containing 0.03 M Tris buffer (pH 7.9), 1 X low3 M glutathione, and 90 mg of lysozyme. The suspension was incubated at 37°C for 45 min with frequent stirring. Subsequent steps for the isolation of this enzyme and other enzyme activities were carried out at 0-4°C except as noted. The cells were then centrifuged at 48,OOOg for 15 min, and the resulting pellet was shocked by suspension in 75 ml of ice-cold 0.01 M Tris-maleate buffer (pH 6.0). This suspension was then treated with deoxyribonuclease at 25°C as previously described and centrifuged at 20,OOOg for 30 min. The resulting supernatant was further centrifuged at 48,OOOg for 30 min. This supernatant, or one prepared by centrifugation at 100,OOOg for 1 hr, was used for DEAE-cellulose chromatography. More activity can be washed from the 20,OOOg pellet by resuspending it in 50 ml of 0.01 M Tris buffer (pH 7.0) and centrifuging at 48,OOOg for 30 min. Some activity, however, always remains in the pellet. This activity can be solubilized by suspension of the pellet in l-2 vol of 0.01 M Tris buffer (pH 7.5) con-
UNDECAPRENYL
PYROPHOSPHATE
FRLCTION
SYNTHETASE
377
NUMBER
1. Chromatography of 48,000g supernatant on DEAE-cellulose in Triton X100. Methods of chromatography and enzyme assay are described in the experimental section. Aliquots of 0.2 ml each were taken for determination of protein concentration and enzyme activity from the 14-ml fractions. The trichloroacetic acid hydrolysis in the assay procedure was carried out at 60°C for 10 min. Closed circles represent A660 nm (protein), and open circles represent cpm observed in the enzymic assay. FIG.
taining 0.01 M MgC12, lyO Triton X-100, and 0.01 M mercaptoethanol. The 48,000g supernatant, containing 405 mg of protein, was applied to a 40 X 2.5 cm DEAE-cellulose column equilibrated with 0.01 M Tris buffer (pH 7.0) containing lro Triton X-100. The protein was eluted from the column with a linear gradient of NaCl (CO.6 M) in the Tris-Triton X-100 buffer, using 750 ml of elutant (Fig. 1). Protein was determined by the method of Lowry el a.l. (18). A precipitate formed during the protein determination due to the presence of Triton X-100 and was removed by centrifugation before determining the optical density at 660 nm. The enzyme activity was measured by the general procedure indicated below. Those fractions containing activity were pooled, concentrated threefold by ultrafiltration, and stored at -20°C. The membrane preparation used as a source of C:s-isoprenyl pyrophosphate phosphatase (2) was obtained from freeze dried !W. Zysodeidficus. Ten grams of cells were treated with lysozyme and deoxyribonuclease according to a previously described procedure (7). The crude broken cell suspension was centrifuged at 27,OOOg for 20 min. The resulting pellets were resuspended in 0.05 M Tris buffer (pH 7.5) and sonicated with a Biosonik II for 5 min while cooled in an ice-salt water slurry. The sonicate was centrifuged at 27,000g for 20 min. The supernatant was t.hen centrifuged at lOO,OOOq
for 1 hr. The resulting pellets were resuspended in 0.05 M Tris buffer (pH 7.5) containing0.1 I/I EDTA, I mM ascorbic acid, and centrifuged at 100,OOOg for 1 hr. This membrane pellet was resuspended in 0.05 M Tris buffer (pH 7.5) containing 1 mM ascorbic acid and used as the source of the Cis-isoprenyl pyrophosphate phosphatase. A second phosphatase activity was prepared from baker’s yeast (15 g) which had been SLW pended at room temperature in 250 ml of lO”x sucrose for 1.5 hr. The yeast were harvested by centrifugation and mixed with approximately 15 g of washed sea sand. Sufficient 0.05 M Tris buffer (pH 7.5) was added to bring the total volume to 40 ml. This mixture was ground at 0°C for 10 min and then centrifuged at 8OOg for 10 min. The supernatant was used as the crude phosphatase preparation from yeast (8). Enzyme assay. The assay solution contained in a final volume of 0.5 ml: 0.21 mM Tris buffer (pH 7.0), 50 pM farnesyl pyrophosphate, 30 SM A3-[lWlisopentenyl pyrophosphate (30,000 dpm), 0.5%. Triton X-100, enzyme, and a variable amount of MgCla depending on the experiment. The reaction mixtures were incubated for 30 min at 30°C. unless otherwise indicated. The reaction products were acid hydrolyzed with 25% trichloroacetic acid at 100°C for 30 min, neutralized with base, and extracted with pet’roleum ether as previously described (7) unless otherwise indicated. A 5-ml
378
KEENAN
AND ALLEN
aliquot of the petroleum ether extract was evaporated to dryness and assayed for radioactivity in toluene-Omnifluor (New England Nuclear) scintillation fluid. Product isotation. The following represents a typical incubation for the formation of radioactive product to be used in the purification and characterization of polyprenyl pyrophosphate. A reaccontaining A3-[1J4C]isopentenyl tion mixture pyrophosphate (187,500 dpm), 0.05 mM farnesyl pyrophosphate, 0.5?& Triton X-100, 0.01 M MgClz, 0.1 M Tris buffer (pH 7.0), and 0.69 mg of DEAEpurified enzyme in a total volume of 3.0 ml was incubated at 37°C for 5 hr. The entire reaction mixture was chromatographed in 0.2 M Tris buffer (pH 7.5) on a Sephadex G-25 column (VO = 14 ml), collecting 5-drop fractions. Aliquots from the column were assayed for radioactivity in a dioxanebased scintillation fluid (19). The polyprenyl pyrophosphate was isolated from the protein-product mixture obtained by Sephadex G-25 chromatography (Fig. 2) by extraction with I-butanol or CHCla-methanol (2: 1, v/v). The solvents were removed from the product by rotary evaporation under vacuum at room temperature to avoid degradation of the product to less polar uncharacterized products. The thin layer radiochromatograms illustrated in Figs. 4,5, and 6 were obtained using a Packard
Model ing at tector on an
7201 Radiochromatogram Scanner recorda chart rate of 0.5 cm/min and full scale deresponse of 300. Mass spectra were obtained AEI-MS30 mass spectrometer. RESULTS
Enzyme Purifccation The profile for elution of protein and enzyme from DEAE-cellulose illustrated in Fig. 1 is essentially the same for a 48,000g supernatant, 100,OOOg supernatant, or a detergent extract of the 20,OOOgpellet. The peak of enzyme activity routinely corresponded to a molarity of 0.35-0.45 M in NaCl in the eluting solvent. DEAE-cellulose chromatography resulted in approximately a threefold purification of the enzyme. If the DEAE-cellulose chromatography was carried out in the absence of Triton X-100, the eluted protein had an enzyme activity less than one qua.rter of that observed when detergent. was included in the eluting solvent. Before evaluation of the enzyme activity, detergent was always added to the enzyme assay mixtures. Attempted purification of the enzyme on hydroxy-
6
40
80 FRACTION
120
160
NUMBER
FIG. 2, Sephadex G-25 chromatography of reaction mixtures after incubation of enzyme with A*-[l-14C]isopenteny1 pyrophosphate with and without farnesyl pyrophosphate. Conditions for this incubation mixture are described in the experimental section. The closed circles represent the observed radioactivity in the presence of 0.05 mM farnesyl pyrophosphate, and the open circles represent radioactivity observed when farnesyl pyrophosphate is omitted from the reaction mixture.
UNDECAPRENYL
PYROPHOSPHATE
apatite, by gradient elution in 0.014.10 M potassium phosphate buffer (pH 7.5) in the presence of 1% Triton X-100, resulted in a marked loss in enzyme activity. Phosphate, however, does not inhibit t’he enzyme activity. Reaction Requirements Incubation of farnesyl pyrophosphate and A3-[lJ4C]isopentenyl pyrophosphat’e with the enzyme isolated from DEAE-cellulose chromatography resulted in the formation of acid labile polyprenyl pyrophosphate. Table I illustrates that product formation was dependent on the presence of farnesyl pyrophosphate and Triton X-100. It is also apparent that the enzyme is only partially inactivated by the ethanol extraction procedure used to remove detergent from the enzyme. Preliminary evidence indicates an apparent divalent cation requirement since the enzyme is completely inhibited by 10, 1, and TABLE RUCTION
Reaction
I
REQUIREMENTS FOR THE SYNTHESIS POLYPRENYL PYROPHOSPHATE~ components
Complete Minus farnesyl phosphate Minus Triton Minus MgC12 Boiled enzyme
pyroX-100
Enzyme
OF
fraction
DEAEcellulose fraction #pm)
Ethanol washed DEAEcellulose fraction (dpm)
13,480 209
4,220 60
13,530 63
77 6,120 45
5 The complete reaction vessel contained the same concentration of components as indicated in the general assay procedure with the addition of M&l* (10 mM) and 0.34 mg of protein except as indicated. Each reaction mixture was incubated at 30°C for 30 min and analyzed as described in the general assay procedure. The ethanol washed DEAE-enzyme was prepared by treatment of the DEAE enzyme with 2 vol of ice-cold ethanol, washing the resulting precipitate twice with 2 vol of ethanol, and resuspending enzyme in 1 vol of 0.01 M Tris buffer (pH 7.0). This procedure removes the Triton X-100 from DEAE-enzyme.
379
SYNTHETASE TABLE
II
ACTIVATION OF ETHANOL WASHED SYNTHBTASE TRITON X-SERIES DETERGENTS~ Detergent Triton Triton Triton Triton None
BY
Radioactivity in product (dpm)
X-45 X-100 X-102 X-114
201 477 478 635 21
0 Each reaction vessel contained the same concentration of components as indicated in the general assay procedure (without magnesium) using 0.12 mg of enzyme and the detergents at a final concentration of 1%. Each reaction mixture was incubated at 30°C for 30 min and analyzed as described in the general assay procedure. The ethanol washed enzyme was prepared as described in Table I.
0.1 mu EDTA in the absence of added magnesium. The inhibition observed at 10 mM EDTA can be overcome by the addition of 20 rnM MgCL. Table II illustrates that several detergents of the Triton X-series will activate the ethanol-washed DEAE-enzyme. Under the same assay conditions, a variety of other detergents, Brij 35 (1.7 %), Brij 58 (1.7 %), Tween 80 (l%), deoxycholate (l%), and sodium dodecyl sulfate (l%), were all ineffective in stimulating the ethanol washed enzyme. Product Isolation The polyprenyl pyrophosphate can be separated from radioactive starting material, A3-[1-14C]isopentenyl pyrophosphate, by chromatography on Sephadex G-25 (Fig. 2). The polyprenyl pyrophosphate cochromatographs with protein emerging in the void volume. Proof that the radioactivity associated with the protein is product specific and not due to nonspecific binding of A3[lJ4C]isopentenyl pyrophosphate is illustrated by the small amount of radioactivity associated with the protein when farnesyl pyrophosphate is omitted from the reaction vessel. Furthermore, the radioactive product associated with the protein can be removed by extractionwitk e&her l-bt&rtol or chloroform-methanol (2: 1, v/v). A eom-
380
KEENAN
AND
parison of the chromatographic properties on thin layer chromatography (Solvent V) of this product (R, 0.47) with A3-[l-‘*Clisopentenyl pyrophosphate (Rf 0.01) indicated that there is no isopcntenyl pyrophosphatc bound to protein. Product
Characterization
The rate of hydrolysis of the polyprenyl pyrophosphate, at 100°C in 17 and 25% trichloroacetic acid, was determined by measuring the formation of petroleum ether soluble radioactivity (Fig. 3). The maximum petroleum ether soluble radioactivity was formed after about 20 min at each concentration of acid and represents about 92 % (25% trichloroacetic acid) of the total radioactivity associated with the product. The radioactive product resulting from this acid hydrolysis chromatographs as essentially one component by thin layer chromatography on silica gel G sheets in solvents IV, V, and VI with Rfs of 0.92, 0.96, and 0.93, respectively. Similarly, acid hydrolysis of the poly-
ALLEN
prenyl pyrophosphate in 25 % trichloroacetate acid at reduced temperature (6O’C) for 10 min renders greater than 90% of the radioactivity as petroleum ether soluble material. The products of this hydrolysis chromatograph on silica gel G sheets as two major components with Rfs 0.92 and 0.83 in Solvent IV. The faster and slower moving components are probably an undecaprenyl hydrocarbon and tertiary alcohol, respectively, as evidenced by similar chromatographic properties observed by Stone and Strominger (20) for the acid hydrolysis products of undecaprenyl pyrophosphate. The polyprenyl pyrophosphate is completely resistant to alkaline hydrolysis in 10% KOH for 1 hr at 100°C. Polyprenyl pyrophosphates synthesized by enzymes from M. lysodeilcticus are resistant to E. coli alkaline phosphatase at 37°C (7, 8). The polyprenyl pyrophosphate isolated here is completely resistant to the action of E. coli alkaline phosphatase at pH 9.2 in the presence or absence of Triton X-100 at 37” and 50°C. The alkaline phos-
6
MINUTES
FIG. 3. The acid lability of the enzymic product at 100°C. The polyprenyl pyrophosphate was prepared by the procedure described in Fig. 2. The protein-product mixture excluded from the Sephadex G-25 was divided into several 0.6-ml aliquots. To each aliquot was added either 0.3 ml or 0.6 ml of 500/e trichloroacetic acid. All the acidified aliquots were then heated in a boiling water bath, and at various periods of time samples were removed, cooled in an ice bath, neutralized with 0.3 ml or 0.6 ml of 5 M NaOH, respectively, and extracted with petroleum ether as in the general enzyme assay. The closed and open circles represent product formation in the presence of 17% and 257, trichloroacetic acid, respectively.
UNDECAPRENYL
PYROPHOSPHATE
SYNTHETASE
381
for the polyprenyl monophosphate (Fig. 4) (20). Cochromatography of the product of the i?f. lysodeilhus membrane treated polyprenyl pyrophosphate with untreated polyprcnyl pyrophosphate is illustrat’ed in Fig. 5. When the polyprenyl monophosphatc was treated with a crude yeast’ homogenate a radioactive component was obt’ained which chromatographed in Solvents IV and V with R/s 0.74 and 0.94, respectively, that correspond to those of undecaprenol isolated and FRONT ORl61N characterized from L. plantarum (Kg. 6AFIG. 4. Chromatogram of polyprenyl monoB).3b All of the observed radioactivity on phosphate obtained by C&s-isoprenyl pyrophoschromatogram A, Fig. 6, was cluted from phate phosphatase treatment of L. plantarum the silica gel, mixed with authentic unpolyprenyl pyrophosphate. The polyprenyl pyrophosphate isolated as described in the experidecaprenol, and subjected to reverse phase mental section was incubated for 5 hr, at 37°C in chromatography in Solvent VII (Fig. 7). 0.6 ml of a solution containing 0.1 ml of M. ZysoA component was observed which codeikticus membrane preparation (1.92 mg), Tris chromatographed with authentic undecbuffer (pH 7.5), 95 rmoles; EDTA, 7 pmoles; aprcnol. The lack of coincidence between Triton X-100, 1%; MgC&, 70 rmoles. The product authentic undecaprenol and all of the radiowas isolated from the reaction mixture by extracact’ivity associated with the enzymatic tion twice with an equal volume of I-butanol. The polyprenol product indicates that there is butanol extract was washed with 0.5 vol of water more than one component present with an and concentrated. The product (approximately 10,000 dpm) was dissolved in ethanol and spotted Rf similar to that of undecaprenol. During on a silica gel G sheet,. The sheet was developed in t’he large scaleisolation of undecaprenol from Solvent V. L. plantarum, we have never detected more than one component (undecaprenol) having phatase is active on the artificial substrate this Rf. It is possible that’ isomeric or longer p-nitrophenyl phosphate under the same conditions. The butanol or chloroform-methanol extracted product has been further charactcrized dures.
by additional chromatographic The product chromatographs
procein
Solvent V with Rf 0.47, J+hich is what is expected for a polyprenyl pyrophosphate3” (20). Treatment of this polyprenyl pyrophosphate with M. lysodeilcticus C&-isoprenyl pyrophosphate phosphatase (2) results in the formation of a radioactive component with an R, 0.60 in Solvent V corresponding to what would be expected 3 (a) Studies by Kenneth Meyer (unpublished work) in this laboratory indicate that the C3&& polyprenyl pyrophosphates synthesized by the polyprenyl pyrophosphate synthetase from M. Zysodeikticus have RJS from 0.2 to 0.34 in the same chromatographic system. (b) Treatment of the pyrophosphates from M. Gas-Go polyprenyl lysocleikticus with the yeast homogenate gave a polyprenol product which chromatographed in solvent IV with an RI of 0.47.
FRONT
ORl6lN
5. Chromatogram of a mixture of L. plantarum polyprenyl pyrophosphate and polyprenyl monophosphate. Polyprenyl monophosphate was prepared from polyprenyl pyrophosphate as described in the legend of Fig. 4. Approximately 8,000 dpm each of polyprenyl monophosphate and polyprenyl pyrophosphate were combined and co-chromatographed on a silica gel G sheet in Solvent V. FIG.
382
KEENAN
AND
II.4 0 0, FRONT
ORIGIN
6. Chromatograms of the products formed by sequential action of Css-isoprenyl pyrophosphate phosphatase and yeast phosphatase on L. FIG.
plantarum polyprenyl pyrophosphate. Polyprenyl pyrophosphate was incubated with membranes from M. lysodeikticus (0.98 mg) as described in Fig. 4. After 5 hr, 120 pmoles MgClz and 1 ml of the yeast preparation were added, and the mixture was incubated an additional 5 hr at 37°C. The product was extracted from the reaction mixture
ALLEN
hydrolyzed by M. lysodeikticus membranes to polyprenyl monophosphate in a manner characteristic of the action of Ce5-isoprenyl pyrophosphate phosphatase on undecaprenyl pyrophosphate (20). Finally, the hydrolytic product resulting from the treatment of polyprenyl monophosphate with yeast homogenate chromatographs primarily as undecaprenol (Figs. 6 and 7). Its chromatographic mobility is similar to that observed for a C&,-polyprenol isolated in a similar manner from a mixture of polyprenyl pyrophosphates produced by the M. lysodeikticus synthetases (8). Although several of the solvent systems used are not highly selective with regard to distinguishing chain length, the chromatographic mobilities of the radioactive alcohol on reverse phase chromatography illustrates cochromatography of the product with undecaprenol. Although the major compo-
with three 5-ml portions of CHCY-methanol, 2:l (v/v), and the extract was washed with 1 ml of water.
The
product
(approximately
5,000
dpm)
was divided into two equal portions and chromatographed on a silica gel G sheet in Solvent (Chromatogram A) and Solvent V (Chromatogram
IV
B).
chain polyprenols are formed that either are not found as free polyprenols following base hydrolysis of L. plantarum or are produced under the unregulated conditions of our in vitro experiments. DISCUSSION We have presented evidence for the isolation of a polyprenyl pyrophosphate synthetase from L. plantarum. The primary enzymic product has been characterized as
undecaprenyl pyrophosphate. The polyprenyl pyrophosphate chromatographs similarly to undecaprenyl pyrophosphate isolated from AI. lysodeikticus (20). The conditions for its acid hydrolysis and t,he chromatograptiic properties of the resulting hydrolysis products are characteristic of undecaprenyl pyrophosphates.
The extracted polyprenyl product has been further characterized by the chromatographic properties of its enzymic hydrolysis products. Polyprenyl pyrophosphate was
9 ORIGIN
FRONT
FIG. 7. Reversephasechromatogramof polyprenol formed by sequential treatment of polyprenyl pyrophosphate with Csh-isoprenyl pyrophosphate phosphatase and yeast phosphatase. The polyprenol (approximately 1,000 dpm) was eluted from the chromatogram illustrated in Fig. 6A and subjected to analytical scale reverse phase thin layer chromatography. Some variation in the RI of a sample has been experienced with reverse phase thin layer chromatography on this scale. Accordingly, authentic undecaprenol (L. plantarum) was added to the enzymatic product (A) prior to chromatography. A second undecaprenol sample (B) was also chromatographed. The plate was visualized with 12, and the labeled portion of the plate was cut into 0.6 X 2 cm bands and counted in toluene scintillation fluid. The unlabeled sample of undecaprenol was visualized with anisaldehyde reagent to confirm the results obtained with 1~.
UNDECAPRENYL
PYROPHOSPHATE
ncnt is most likely undecaprenol, the prescnce of related compounds such as isomeric or longer chain polyprenols is possible. It should be pointed out, however, that in vivo (14) and in vitro (9) stud& on the biosynthesis of polyprenols indicate that undecaprenol is the major and highest molecular weight polyprenol synthesized by L. plantarum.
Scphadcx G-25 chromatography of the complete reaction mixture permits a rapid and complete separation of the enzymic product from starting materials. Although the product accompanies the protein in the void volume of the column, no definitive evidence has been presented t,o indicate if any interaction between protein and product exists. Previous work of Kurokawa et al. (8) indicates that long chain polyprenyl pyrophosphates are excluded from Sephadex G-25 in the absenceof protein. Soncovalent associat’ion of polyprenyl pyrophosphat’es with protein has previously been described by Allen et al. (7) for the C&C40 polyprcnyl pyrophosphate synthet’ase from M. lysodeikticus, Oster and West (21) and Ogura et al. (22) for gcranylgeranyl pyrophosphate synthetase, and Rilling (23) for presqualene pgrophosphate synthetase. The enzyme, like other polyprenyl pyrophosphate synthetases, demonstrates a requirement for farnesyl pyrophosphatr and divalent cation. In addition, enzyme activ y is also dependent on the addition of detergent. The nonionic detergents of the Triton X-series are effective, whereas, other nonionic and ionic detergents tested do not’ activate the enzJTme.This requirement for the presence of detergent’ is unique among t,he polyprenyl pyrophosphate synthetases thus far described, since neither the C&Z4,, polyprenyl pyrophosphatc synthetase (7, 8) nor the undecaprenyl pyrophosphate synthcttases (6, 8, 9) require the presence of detergent for cnzymic activit’y. Studies are continuing t’o further charact’erize both the undccaprcnyl pyrophosphate and its synthctase. ACKNOWLEDGMENTS We are grateful to Dr. Roy out the mass spectral analyses nol.
King for carrying of the undecapre-
SYNTHETASE
383
REFERENCES 1. ROTHFIFLD, L., AND ROMEO, 1). (1971) Bacteriol. Rev. 36, 14-38. 2. GOLDMAN, R., BND STROMINGER, J. L. (1972) J. Biol. Chem. 247, 5116-5122. 3. HIGASHI, Y., SIEWERT, G., AND STROMINGER, J. L. (1970) J. Biol. Chena. 246, 3683-3690. 4. WILLOUGHBY, E., HIGASHI, Y., AND STROMINGER, J. L. (1972) J. Biol. Chem. 247, 51135115. 5. THORNE, K. J. I., AND BARKER, D. C. (1971) Biochem. J. 122, 45P46P. 6. CHRISTENSON, J. G., GROSS, S. K., AND ROBBINS, P. W. (1969) J. Biol. Chem. 244, 54365439. 7. ALLEN, C. M., AL~ORTH, W., MACCR~E, A., AND BLOCH, K. (1967) J. Biol. Chem. 242, 1895-1902. 8. KUROK~TVA, T., OGURA, K., AND &TO, S. (1971) Biochem. Biophys. Res. Commun. 46, 251-257. 9. DURR, I. F., END HABBAL, M. Z. (1972) Biothem. J. 127, 345-349. 10. POPJAK, G., CORNFORTH, J. W., CORNFORTH, H. H., R~H~GE, R., AND GOODMSN, D. S. (1962) J. Biol. Chem. 237, 56-61. 11. HOLLOWBY, P. W., AND POPJaK, G. (1967) Biochem. J. 104, 57-70. 12. YUAN, C., AND BLOCH, K. (1959) J. Biol. Chem. 234, 2605-2608. 13. DUGAN, R. E., Rasson-, E., AND PORTER, J. W. (1968) Anal. Biochem. 22,249-259. 14. GOUGH, D. P., KIRBY, A. L., RICHARDS, J. B., AND HEMMING, F. W. (1970) Biochem. J. 118, 167-170. 15. MCSTVEENEY, G. P. (1965) J. Chromatogr. 17, 183-185. 16. &EGGS, H. R., WRIGHT, L. D., CRESSON, E. L., &1acRa~, G. D. E., HOFFMAN, C. H., WOLF, D. E., .LND FOLI~ERS, K. (1956) J. Bacterial. 72, 519-524. 17. HA~EL~L, Z., AND DURR, I. F. (1969) J. Bacterial. 100, 1409-1410. 18. Lowax-, 0. H., ROSERROUGH, N. J., F.~RR, 8. L., AND RANDALL, It. R. (1951) J. Biol. Chem. 193, 265-275. 19. ALLEN, C. M. (1972) Biochemistry 11, 21542160. 20. STONE, K. J., AND STROMINGER, J. L. (1972) J. Biol. Chem. 247, 5107-5112. 21. OSTER, M. O., AND WEST, C. A. (1968) Arch. Biochem. Biophys. 12’7, 112-123. 22. OGURA, K., SHINKA, T., AND SETO, S. (1972) J. Biochem. 72, 1101-1108. 23. RILLING, 1%. C. (1970) J. Lipid Res. 11, 480485.