Proc. Nat. Acad. Sci. USA Vol. 69, No. 5, pp. 1287-1289, May 1972

Inhibition of Sterol Biosynthesis by Bacitracin (mevalonic acid/CQ-isopentenyl pyrophosphate/C15-farnesyl pyrophosphate) K. JOHN STONE* AND JACK L. STROMINGERt Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Contributed by Jack L. Strominger, March 14 1972

ABSTRACT Bacitracin is an inhibitor of the biosyn- muslin eliminated floating particles of fat from the enzyme thesis of squalene and sterols from mevalonic acid, C5- solution enzyme). In some the isopentenyl pyrophosphate, or C15-farnesyl pyrophosphate (Sio experiments, S10 enzyme catalyzed by preparations from rat liver. The antibiotic was centrifuged at 105,000 X g for 90 min to remove micro- is active at extremely low ratios of antibiotic to substrate. somal particles, and this supernatant is referred to as the Sc1t The mechanism of inhibition appears to be the formation enzyme. of a complex between bacitracin, divalent cation, and C15- Bacitracin was generously provided by the Upjohn Co., farnesyl pyrophosphate and other isoprenyl pyrophos- phates. It is similar to the formation of the complex with Kalamazoo, Mich. [2-14C]3RS-mevalonic acid was obtained C55-isopropenyl pyrophosphate in microbial systems. The from New England Nuclear Co., [SH]farnesyl pyrophosphate toxicity of bacitracin for animal cells could be due in part and [2-'4C]isopentenyl pyrophosphate were generous gifts to the formation of these complexes. from Dr. Konrad Bloch. The Enzyme incubations (about 0.5-ml samples) were termi- antibacterial properties of bacitracin result, in part, from nated by in a water bath for 1 an inhibition of the heating boiling min. They were enzyme, C05-isoprenyl pyrophosphate then extracted three times with an volume of phosphatase (EC 2.5.1.1), which is responsible for the re- equal diethyl generation of ether. The ether extracts were filtered through a bed of about C56-isoprenyl phosphate, an essential carrier in 1 ml of basic alumina (Woelm) and washed with a the biosynthesis of bacterial cell walls (1, 2). In a preceding through the further 5 ml of ether. The alumina filtration ensured that all of paper (3), mechanism of inhibition has been shown to be the substrates were removed from the ether extracts. the formation of a complex between bacitracin, a divalent The cation, and the ether eluate was evaporated to about 0.05 ml and diluted to C55-isoprenyl pyrophosphate. Thus, bacitracin 1 ml with toluene. A portion was then assayed for radioac- acts by complexing an essential substrate rather than by tivity. inactivating an enzyme. The proposed mechanism of complex formation suggested the possibility that other pyrophos- RESULTS phates, structurally related to C05-isoprenyl phosphate, might Inhibition by bacitracin of the synthesis of squalene and also complex with bacitracin and that the antibiotic would sterols from various precursors inhibit enzymatic reactions involving these compounds. The biosynthesis of squalene and sterols from mevalonic acid, The biosynthesis of squalene and sterols from mevalonic isopentenyl pyrophosphate, and dimethylallyl pyrophosphate acid involves a number of intermediates amongst which di- is well established (see for example ref. 5). In this complex methylallyl pyrophosphate (Cr-isoprenyl pyrophosphate), reaction sequence, squalene and later products are soluble in geranyl pyrophosphate (trans-Clo-isoprenyl pyrophosphate), diethyl ether while squalene precursors are water soluble and and farnesyl pyrophosphate (all-trans-C16-isoprenyl pyrophos- are strongly adsorbed by alumina. Utilization of these prop- phate) differ from bacterial C05-isoprenyl pyrophosphate only erties allows a simple evaluation of the squalene plus sterol in the length and stereochemistry of the isoprenoid chain. In synthesizing capacity of an enzyme mixture. the present paper, bacitracin is shown to be an effective The conversion of 3RS-mevalonic acid to squalene was inhibitor of this biosynthetic sequence in vitro. The mechanism found to be completely inhibited at a bacitracin concentration of inhibition appears to be identical with that of the bacterial of 1 mol per 2 mol of mevalonic acid added. As only the 3R- system, and formation of a complex between farnesyl pyro- mevalonic acid isomer is used as substrate (6), this is equiva- phosphate, bacitracin, and a divalent cation has been lent to a molar ratio of inhibitor to substrate of 1 to 1. The demonstrated. conversions of isopentenyl pyrophosphate to squalene and MATERIALS AND METHODS farnesyl pyrophosphate to squalene were also inhibited by Enzyme solutions were prepared as described by Popjak (4). bacitracin, although these were less sensitive (Fig. 1). The de- Minced livers from young male Sprague-Dawley rats (100- creased sensitivity of the system to bacitracin from mevalonic 120 g) were gently homogenized with a Potter-Elvehjem acid through isopentenyl pyrophosphate to farnesyl pyro- homogenizer in 2.5 volumes of buffer containing 0.1 M Tris- phosphate suggests that more than one of the intermediate re- HC1, 5 mM MgCl2, 2 mM MnCl2, and 30 mM nicotinamide at actions is sensitive to the antibiotic. pH 7.5. Centrifugation for 30 min at 15,000 X g removed Inhibition by bacitracin of the biosynthesis of nuclei, cell debris, and mitochondria, and filtration through isoprenoid pyrophosphate from mevalonic acid and isopentenyl pyrophosphate * Present address: Roche Research Laboratory, Department of An experiment that shows that other reactions in the bio- Biochemistry, Liverpool University, Liverpool, England. synthetic sequence are also inhibited by bacitracin is illus- t To whom reprint requests should be sent. trated in Fig. 2. In this experiment, the absence of micro- 1287 Downloaded by guest on September 26, 2021 1288 Biochemistry: Stone and Strominger Proc. Nat. Acad. Sci. USA 69 (iI97e) somal particles ddes not allow the formation of squalene, and the immediate precursor, farnesyl pyrophosphate, is the major product (7, 8). Hydrolysis of isoprenyl pyrophosphates with alkaline phosphatase liberates free isoprenol, which is extractable by ether and eluted from alumina. The radioac- tivity eluted from the alumina, thus, gives a measure of the ~40 amount of geranyl pyrophosphate and farnqsyl pyrophosphate formed. The observation that, at high bacitracin concentra- K ~~~~~~LipidReleased by Phosphotose tions and with isopentenyl pyrophosphate as substrate, Jittle radioactivity is eluted from the alumina column shows that little or no isopentetiol is isolated by the above procedure. The 20- small amounts of radioactivity that can be extracted from these incubations before treatment with alkaline phosphatase Squalene + Sterois is undoubtedly due to contamination of the enzyme with small 02 0.4 06 Q8 amounts of residual microsomal fragments. Nevertheless, nmol Bacitracin/nmol Substrate this "background" radioactivity is small compared with that eluted after phosphatase treatment, and a strong inhibitory FIG. 2. Percent products formed from mevalonic acid or isopentenyl pyrophosphate in the presence of Sio5 enzyme and effect of bacitracin on the biosynthesis of isoprenyl pyro- various concentrations of bacitracin. Incubation mixtures con- phosphate is apparent. tained 2.5 mg ATP, bacitracin at the appropriate concentration, Inhibition by bacitracin of the dephosphorylation of 0.5 ml of Soi5 enzyme, and either 320 nmol of [2-14C]mevalonic farnesyl pyrophosphate by Escherichia coli acid, or 74 nmol of [14C]isopentenyl pyrophosphate, each with the phosphomonoesterase specific activities given in Fig. 1. The final incubation volume was 0.6 ml. Incubations were terminated by boiling for 1 min and It has been shown that E. coli phosphomonoesterase catalyzes analyzed as in Methods. The remaining aqueous extract was the dephosphorylation of C06-isoprenyl pyrophosphate and warmed at 60° for 30 min to remove all traces of ether, the pH that this dephosphorylation is inhibited by bacitracin (2). In was adjusted to 8.2 with 0.5 M Tris buffer, and the final mixture the present experiment, farnesyl pyrophosphate has been was hydrolyzed at 370 for 60 min with 7.5 Units of Escherichia used as a substrate for the E. coli phosphomonoesterase. Its coli alkaline phosphatase. This second enzyme reaction was dephosphorylation was similarly inhibited by bacitracin, but terminated and analyzed for radioactive products as in Methods. the hydrolysis of p-nitrophenyl phosphate catalyzed by the A-A, percent phosphatase-labile products from isopentenyl E. coli enzyme was not significantly altered by the antibiotic pyrophosphate; O-O, percent phosphatase-labile products (Fig. 3). This result suggests that the bacitracin interacts from mevalonic acid; A-A, percent squalene and sterol from with the substrate rather than with the enzyme. isopentenyl pyrophosphate; *-*, percent squalene and sterol from mevalonic acid.

Direct demonstration of formation of a complex between bacitracin and farnesyl pyrophosphate By the method of Hummel and.Dryer (9), ['H farnesyl pyrophosphate was filtered through a column of Sephadex G-25 in buffer containing Mg++. The addition of bacitracin during the period when the elution of 'adioactivity was con- stant resulted in a peak of radioactivity followed by a trough (Fig. 4). This pattern is indicative of the formation of a complex and is the mirror image of the effect observed with ["2PlC,,- isoprenyl pyrophosphate (3). For farnesyl pyrophosphate, which can be kept in aqueous solution without the addition of a detergent, no micelle formation occurs and the pattern ob- nmol Bacitracin/nmol Substrate tained resembles that obtained in other binding studies (9). the was in the absence of FIG. on of When experiment repeated Mg++, 1. Effects of bacitracin the biosynthesis squalene acid and sterols by Slo enzyme from various precursors. Incubation but in the presence of ethylene diamine tetraacetic mixtures contained 3 mM ATP, 1 mM NADP, and 3 mM (EDTA), the positive and negative peaks of radioactivity glucose-6-phosphate. To this was added either 800 nmol of were virtually absent, indicating a requirement for metal ions [2-14013RS-mevalonic acid (4.7 Ci/mol), or 20.9 nmol of ['H]- in the binding mechanism. farnesyl pyrophosphate (35 Ci/mol), or 74 nmol of [14C]iso- Under the conditions of these experiments, identical binding pentenyl pyrophosphate (1 Ci/mol). Bacitracin was added at curves were obtained with ['HIfarnesyl pyrophosphate and the final concentration indicated, and the incubation mixtures bacitracin in the presence of Zn++, Cd++, Mn++, and Mg++. were made up to 0.6 ml by the addition of 0.5 ml Sio enzyme con- With ['4C]isopentenyl pyrophosphate, the binding appeared taining 16.2 mg of protein. After incubation at 370 for 30 min, the to be much weaker. A small peak and trough were observed in incubations were terminated and analyzed (see Methods). Data the presence of Zn++ and Cd++ but not in the presence of are expressed as percent of radioactivity incorporated in the was not bound incubation mixture from which bacitracin was omitted. For meva- Mn++ or Mg++. "P-inorganic pyrophosphate lonic acid, A- A, 350,000 cpm were present in this control; at all in the presence of Zn++, Mn++, or Mg++ and was very 232,000 cpm for farnesyl pyrophosphate, O-O; and 30,400 cpm weakly bound (if it was bound at all) in the presence of Cd++. for isopentenyl pyrophosphate, *-*. With all substrates, binding was greatly diminished by addi- Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Sterol Biosynthesis and Bacitracin 1289

tion of EDTA. These qualitative experiments should -be regarded as preliminary ones, and efforts are presently under Bocitracin Added A way to measure the binding constants of CM-isoprenyl pyro- phosphate, farnesyl pyrophosphate, isopentenyl pyrophos- 40_ phate, and inorganic pyrophosphate in the presence of various metal ions, as well as to study the structural features of the bacitracin molecule that are required for binding (14). DISCUSSION 20- rn It has been suggested from the known mechanism of action I of bacitracin that this antibiotic should bind to isoprenoid IlIze11 pyrophosphates structurally related to pyro- .)11. C65-isoprenyl R) phosphate. This hypothesis has been tested and found to be 44 correct. The mechanism of binding appears to be identical for 1. farnesyl pyrophosphate and Cm-isoprenyl pyrophosphate, and Z divalent cations are required in each case. a 40_ The finding that bacitracin binds to isoprenyl pyrophos- phates with trans isoprenoid units at the a-terminal end of the molecule (geranyl pyrophosphate and farnesyl pyrophosphate have trans a-units, C55-isoprenyl pyrophosphate has a cis a-unit) indicates that it specifically interferes with all reac- 20 tions involving isoprenoid pyrophosphates. One might ex- pect, therefore, that, in addition to sterol biosynthesis, the biosynthesis of isoprenoid quinones, involving C45-isoprenyl i and C50-isoprenyl pyrophosphates (10), and the biosynthesis of dolichol and dolichyl phosphate, which are presumably 0. 40 80 120 formed by successive additions of isopentenyl pyrophosphate Fraction Number to isoprenyl pyrophosphates and which are required for FIG. 4. Complex formation of bacitracin with [3H]farnesy mannose and glucose transfer (11, 12), would be inhibited in pyrophosphate. (A) The radioactive lipid (4.4 X 106 cpm) in 6.5 animal cells. Inhibition of these important biosynthetic ml of 0.1 M Tris buffer (pH 7.5) containing 20 mM MgCl2 was systems could explain, at least in part, the toxicity of baci- applied to a column of Sephadex G-25 (1.4 ml in a 1-ml graduated tracin for animal tissues. Conceivably, at subtoxic concentra- pipet). Fractions of 40,Ul were collected in a micropipet every 1.5 tions, bacitracin could be a useful hypocholesteremic agent. min, and the entire fraction was counted. Bacitracin (7.0 mg dissolved in 40 ul of the radioactive solution) was added to the top 1OOr of the column at the time indicated, and elution was continued. (B) lb The same experiment was done except that MgCl2 was omitted EDTA at a ! 80so from the radioactive buffer-substrate mixture, and a , ^ final concentration of 40 mM was used. q9 60I- Supported by research grants from the U.S. Public Health % 40 Service (Al-09152 and AM-13230) and the National Science Foundation (GB-29747X). We thank Miss Eileen Willoughby for

2I0 0 technical assistance in some of these experiments. Os,. 20 1. Anderson, J. S., Meadow, P. M., Haskin, M. A. & Stromin- ...... ger, J. L. (1966) Arch. Biochem. Biophys. 116, 487-515. r. a- 5 i 25 2. Siewert, G. & Strominger, J. L. (1967) Proc. Nat. Acad. Sci. Bacitracin Added, mg/ml USA 57, 767-773. 3. Stone, K. J. & Strominger, J. L. (1971) Proc. Nat. Acad. FIG. 3. Effect of bacitracin on the hydrolysis of farnesyl Sci. USA 68, 3223-3227. pyrophosphate and p-nitrophenyl phosphate by E. coli alkaline 4. Popjak, G. (1969) Methods Enzymol. 15, 393-454. phosphatase. Incubations for the hydrolysis of farnesyl pyrophos- 5. Clayton, R. B., ed. (1969) Methods Enzymol. 15, p. 903. 6. Cornforth, J. W., Cornforth, R. H. & Popjak, G. (1962) phate contained lmol10 Tris buffer (pH 8.2), 1.5 Asmol Mg++, Tetrahedron 18, 1351-1354. 0.35 Units of enzyme, 20,000 cpm of farnesyl pyrophosphate 7. Goodman, De. W. S. & Popjak, G. (1960) J. Lipid Res. 1, (about 1.5 nmol), and bacitracin to a final volume of 0.25 ml. 286-300. After incubation at 370 for 10 min, samples were boiled, diluted 8. Popjak, G. (1969) Methods Enzymol. 15, 393-454. with 2,u of unlabeled farnesol, extracted with ether, filtered 9. Hummel, J. P. & Dreyer, W. J. (1962) Biochim. Biophys. through alumina, and assayed for radioactivity. Incubations for Acta 63, 530-532. the hydrolysis of p-nitrophenyl phosphate contained 2.5,umol of 10. Raman, T. S., Rudney, H. & Buzzelli, N. K. (1969) Arch. p-nitrophenyl phosphate, 1l0.umol Tris buffer (pH 8.2), 1.5 ,Imol of Biochem. Biophys. 130, 164-174. P. F. W. Mg++, 0.35 Units of enzyme, and bacitracin to a final volume of 11. Richards, J. B., Evans, J. & Hemming, (1971) 0.25 ml. at 370 for 10 the absorbance of the Biochem. J. 124, 957-959. After incubation min 12. Behrens, N. H., Parodi, A. J. & Leloir, L. F. (1971) Proc. reaction mixture was measured at 420 nm. The percent reaction Nat. Acad. Sci. USA 68, 2857-2860. was calculated assuming a molar extinction coefficient for 13. Malamy, M. & Horecker, B. L. (1966) Methods Enzymol. p-nitrophenol of 1.32 X 104 (13). O-O, farnesyl pyrophos- 9, 639-642. phate; A-iA, p-nitrophenyl phosphate. 14. Storm, D. & Stone, K. J. (1972) Fed. Proc. 31, 910. Downloaded by guest on September 26, 2021