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Cerulenin resistance in a cerulenin-producing fungus
ARCHIVES OF BKXHEMISTRY
AND BIOPHYSICS
Vol. 197, No. 1, October 1, pp. 30-35, 1979
Cerulenin
Resistance
in a Cerulenin-Producing
Fungus
Isolation of Cerulenin Insensitive Fatty Acid Synthetase AKIHIKO *institute
KAWAGUCHI,” HIROSHI TOMODA,* SHIGENOBU OKUDA,* JUICHI AWAYA,? AND SATOSHI &!IURAf
of Applied Microbiology, The University of Tokyo, Tokyo 113, and tThe Kitusuto School of Phm-maceutical Science, Kitasato University, Tokyo 108, Japan
Znstitute and
Received March 14, 1979; revised April 30, 1979 Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium cue&ens, is a potent inhibitor of fatty acid synthetase systems. This antibiotic specifically blocks the activity of P-ketoacyl thioester synthetase (condensing enzyme). The mechanism of the resistance of C. eaerulens to cerulenin was investigated. The rate of growth in medium containing up to 100 pg/ml cerulenin was as rapid as that in cerulenin-free medium. At a cerulenin concentration of 300 pg/ml, the rate of growth was still more than half that of the control. The addition of cerulenin (200 pg/ml) to a culture of growing cells has almost no effect on the incorporation of [‘%]acetate into cellular lipids. Fatty acid synthetase was purified from C. caerulens to homogeneity. Properties of this fatty acid synthetase were almost the same as those of yeast fatty acid synthetase except for the sensitivity to cerulenin. C. cue&ens synthetase is much less sensitive to cerulenin than fatty acid synthetases from other sources. These findings suggested that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus.
Cerulenin is an antifungal antibiotic isolated from a culture filtrate of the fungus Cephalosporium caerulens (1) and has the structure (2R)(3S)-2,3-epoxy-4-oxo-7,10dodecadienoylamide (2).l This antibiotic is a potent and noncompetitive inhibitor of fatty acid synthetase systems isolated from various microorganisms and from rat liver (3, 4). Low concentrations of cerulenin inhibit fatty acid synthetases of all known types, whether they are multienzyme complexes or nonaggregated systems. Further studies of the fatty acid synthetases purified from several organisms indicated that cerulenin specifically blocks the activity of P-ketoacyl thioester synthetase (condensing enzyme) (3, 4). Such a specificity has been useful for many investigators in studies of lipid metabolism and membrane biogenesis (5). We have investigated the mechanism of the resistance of C. caerulens to cerulenin.
We have already reported that the cerulenin producing strain, C. caerulens KF-140, was more resistant to cerulenin than the other species of the same genus, Cephalosporium (6). In this work, we purified fatty acid synthetase from C. caerulens and found that this synthetase was much less sensitive to cerulenin than fatty acid synthetases from other sources. This insensitivity of the enzyme to cerulenin may be responsible for the protection of C. caerulens from inhibitory effects of this antibiotic. MATERIALS
Organism and chemicals. Cephalosporium caerulens KF-140 was grown at 27°C with shaking in a medium containing 30 g of glycerol, 10 g of glucose, 5 g of peptone, and 2 g of NaCl per liter. Growth was recorded as the inerease in the absorbance at 660 nm. Cerulenin was prepared according to the method described previously (6). Sodium [2-%]acetate and [2-“C]malonyl-CoA were purchased from New England Nuclear, Boston; acetyl-CoA and malonyl-CoA were from P-L Biochemicals Inc., Milwaukee, Wiscon-
1 The absolute configuration of epoxide is (2R)(3S) instead of (2S)(3R) as previously reported (1). 0003-9361/79/110030-06$02.00/o
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
30
ISOLATION
OF CERULENIN-INSENSITIVE
FATTY ACID SYNTHETASE
31
sin; NADPH and NADH were from Oriental Yeast Co., Tokyo. All other reagents and solvents were of analytical grade. Microbial assay of cerulenin. The amount of cerulenin was determined by microbial assay employing Candida albicans KF-1 as the test microorganism according to the method reported previously (6). Incorporation of [%‘]acetate into lipids in vivo. C. cue&ens cells, which were grown at 27’C for 6, 24,
and 48 h, respectively, were centrifuged, and resuspended in the growth medium. The turbidity of each cell suspension was adjusted to 0.2 of the absorbance at 660 nm. Aqueous cerulenin solution (0.1 ml) was added to 0.8-ml portions of cell suspensions and the mixtures were incubated at 27°C for 10 min. Subsequently, 0.1 ml of [Wlacetate (0.2 &i) was added to each mixture and incubation was continued for a further 15 min. One milliliter of 10% trichloroacetic acid was added and the cells were washed twice with 5% trichloroacetic acid. Lipids were extracted from the cells with chloroform-methanol (2:1, v/v). Samples of the chloroform-methanol extracts were evaporated to dryness and the radioactivity counted with a lipid scintillation counter. Preparation
of cell-free extracts from C. cue&ens.
Ten grams of C. caerulens cells was suspended in 20 ml of 0.1 M potassium phosphate buffer (pH 7.0) and disrupted for 2 min with 15 g of glass beads in a Braun homogenizer. Cell debris was removed by centrifugation at 10,OOOgfor 5 min and the supernatant was centrifuged again at 80,OOOgfor 1 h. The resulting supernatants were used as the cell-free extracts. Fatty acid synthetase assay. Fatty acid synthetase activity was assayed spectrophotometrically according to the method described previously (7). Unless otherwise stated, assays were carried out at 37°C in mixtures containing 0.1 M potassium phosphate buffer (pH 7.15), 5 mM dithiothreitol, 200 pM NADPH, 50 pM acetyl-CoA, 40 pM malonyl-CoA, 100 pg bovine serum albumin, and enzyme protein in a total volume of 0.5 ml. One unit of activity is defined as the amount of enzyme required to incorporate 1 nmol of malonyl-CoA per minute into fatty acids. Purification offatty acid synthetase. All steps were performed at 0-4°C. Protein was determined by the
FIG. 1. Agarose gel electrophoresis of C. caerulens fatty acid synthetase. Gel electrophoresis on 1% agarose was done according to the method previously described (7). After the gels were destained, they were scanned with a Fujiox (Tokyo, Japan) densitometer. method of Bradford (8). All buffers contained 10 mM 2-mercaptoethanol and 1 mM EDTA, unless otherwise stated. For the preparation of fatty acid synthetase, C. cue&ens was grown to the mid-log phase, for 24 h. Fifty-four grams of frozen cells was thawed in 216 ml of 0.1 M potassium phosphate buffer (pH 7.0). The cells were broken by passage through a French pressure cell operated at 20,000 psi and the disrupted cells were centrifuged at 25,000g for 20 min. The protein concentration of the resulting supernatant was adjusted to 10 mg/ml by adding 0.1 M potassium phosphate buffer (pH 7.0) and the diluted supernatant was brought to 30% saturation with ammonium sulfate. After stirring for 15 min, the precipitate was removed by centrifugation at 25,000g for 20 min and discarded. The supernatant was brought to 50% saturation with ammonium sulfate, stirred for 15 min, and centrifuged at 25,000g for 20 min. The precipitate was dissolved in 18 ml of 0.05 M potassium phosphate buffer (pH 7.0) and dialyzed against 0.05 M potassium phosphate buffer (pH 7.0) for 3 h. The dialyzed solution was applied to a DEAE-cellulose column (2.4 x 20 cm) that had previously been equilibrated with 0.05 M potassium phosphate buffer (pH 7.0). The enzyme was eluted by a linear gradient from 0.05 to 0.25 M potassium phosphate, pH 7.0 (600 ml). The major fractions containing
TABLE I PURIFICATIONOF FATTY ACID SYNTHETASEFROMC. cue&ens
Fraction
Volume (ml)
Crude extract (NH&SOI, 30-50% DEAE-cellulose Sepharose 6B
280 18 120 8.5
Total activity (unit) 29300 11600 7350 1620
Total protein (mg)
Specific activity (unit/mg)
Yield (%)
6020 1190 58.9 3.0
4.87 9.75 125 541
100 39.5 25.0 5.5
32
XAWAGUCHI
enzyme activity were brought to 50% saturation with ammonium sulfate and centrifuged at 25,000g for 20 min. The precipitate was dissolved in 2 ml of 0.25 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1 mM dithiothreitol instead of 10 mM 2mercaptoethanol, and applied to a Sepharose 6B column (1.7 x 50 cm) that had been equilibrated with the same buffer. The fractions containing enzyme activity were combined and concentrated with a Diaflo apparatus with an XM-50 membrane. The results of this purification are summarized in Table I. After the final purification step the synthetase was homogenous, as judged by the appearance of a single band when subjected to electrophoresis on 1% agarose gels (Fig. 1). RESULTS
Effect of Cerulenin on the Growth of C. caerulens A previous paper from our laboratory reported that the growth of C. caerulens reached the stationary phase after about 50 h of cultivation (6). Cerulenin was detected in the culture filtrate after 12 h of cultivation and the amount of cerulenin reached a maximum (about 250 pg/ml) at 48-50 h. To reveal the cerulenin resistance of C. caerulens, growth of this fungus in medium supplemented with cerulenin was carried out and the results are shown in Fig. 2. Early (Fig. 2A) and late (Fig. 2B) logphase cells were transferred to the medium supplemented with cerulenin and the growth was followed by measuring the absorbance at 660 nm. Both the length of the lag phase and the rate of the growth were slightly affected by the age of the inoculum but the
ET AL.
0
100 CERULENIN (pglml )
200
FIG. 3. Cerulenin effect on the incorporation of [14C]acetate into the lipid fraction of C. caerulens. See Materials and Methods for details. Cells grown for 6 (+I, 24 (O), and 48 h (A) were used for experiments. Results are expressed as percentages of 14Cincorporation relative to experiments in which cell suspensions were treated without cerulenin.
growth patterns were essentially the same. The growth rate in the medium containing up to 100 pg/ml cerulenin was as rapid as that in cerulenin-free medium and even with 300 pg/ml cerulenin the growth rate was more than half the rate in the controls. Since the growth of cerulenin sensitive organisms was stopped completely at cerulenin concentration of 100 pg/ml (5), the above results suggested that C. caerulens exhibited a high resistance to cerulenin. Malik and Vining (12) studied the chloramphenicol resistance of chloramphenicolproducing Streptomyces and observed that late log-phase cells showed higher resistance to chloramphenicol than early logphase cells. To explain these observations, they postulated that membrane permeability for the antibiotic changed after the exposure to chloramphenicol. In the case of C. caerulens, it is hard to consider such changes of membrane permeability after exposure to cerulenin, because the growth patterns were not dependent on the age of the inoculum. Effect of Cerulenin on Lipid Synthesis
FIG. 2. Cerulenin effect on the growth of C. caerulens. Cells grown for 8 (A) and 48 h (B) were inoculated into L-tubes, each containing 5 ml of medium supplemented with various concentrations of cerulenin (numbers in parentheses indicate pg/ml). See Materials and Methods for details.
The effect of cerulenin on the incorporation of [14C]acetate into cellular lipids was studied with a growing of C. caerulens (Fig. 3). Growing cultures of three different phases were transferred to fresh medium
ISOLATION
OF CERULENIN-INSENSITIVE
containing [14C]acetate and cerulenin, and the incorporation of acetate into the lipid fractions was measured. Many investigators have confirmed that cerulenin specifically interferes with the incorporation of acetate into lipids in a variety of yeasts, fungi, and bacteria (9-11). In these cerulenin sensitive organisms, 100 pg/ml cerulenin completely inhibits the incorporation of labeled precursor into lipid fractions. On the contrary, lipid synthesis in C. caerulens was inhibited only slightly by cerulenin. The enzyme system for lipid synthesis was still active even at a concentration of 200 @g/ml cerulenin. These results were also confirmed by the experiments with cell-free extracts of C. caerulens. Cell-free extracts prepared from C. caerwlens cells of both mid- and late log phases retained the activity of fatty acid synthesis from [2-14C]malonyl-CoA up to 400 pg/ml cerulenin. Cerulenin was found to retain almost full antibiotic activity during a 30-min incubation with growing cell suspensions and with cell-free extracts. From these results we can rule out the possibility that cerulenin was inactivated during the above reaction of fatty acid synthesis. Since the principal target of cerulenin has been established to be fatty acid synthesis, it is reasonable to consider that the fatty acid synthetase system of this fungus is resistance to cerulenin. Putification and Properties Synthetase
FATTY
ACID
33
SYNTHETASE
for reduced pyridine nucleotide(s). It was found that the stereospecificity of enoyl reductase was dependent on the source of the enzyme; rat liver enzyme was A-specific and enzymes from Brevibacterium ammoniagenes and yeast were B-specific (13-15). The stereospecificity of the fatty acid synthetase from C. caerulens was also determined by the same technique as reported previously (13). Both /3-ketoacyl reductase and enoyl reductase were B-specific. The properties of C. caerulens fatty acid synthetase are similar to those of yeast fatty acid synthetase. Inhibition
of Fatty Acid Synthetases
by
Cerulenin Figure 4 shows the effects of cerulenin on fatty acid synthetases purified from yeast, B. ammoniagenes and C. caerulens, and also on alcohol dehydrogenase from yeast. Cerulenin inhibition of yeast fatty acid synthetase was complete with 5 pglml. Fatty acid synthetase of B. ammoniagenes was as sensitive to cerulenin as yeast synthetase. Vance et al. (4) also reported that fatty acid synthetases of Euglena gracilis, Corynebacterium diphutheriae, and Mycobacterium phlei were inhibited by cerulenin to almost the same extent. On the contrary,
of Fatty Acid
To confirm the conclusion that fatty acid synthetase itself is resistant to cerulenin, we purified fatty acid synthetase from C. caerulens (Table I) and obtained a homogeneous preparation of this enzyme (Fig. 1). The pH optimum of the enzyme activity was 7.15. The apparent K, values for the three substrates were: Malonyl-CoA, 9.1 PM; acetyl-CoA, 9.3 PM; and NADPH, 14 PM. The molecular weight, estimated by chromatography on Sepharose 6B, was estimated to be 1.2 x lo6 in three trials. Under the standard assay conditions, 41% of the fatty acids produced is palmitate and 59% stearate. Recently we determined the stereospecificities of P-ketoacyl and enoyl reductases (partial reactions of fatty acid synthetase)
” 10
50
100
200 CERULENIN
300
400
(pglml)
FIG. 4. Percentage inhibition of fatty acid synthetases and yeast alcohol dehydrogenase by cerulenin. The symbols in the graph are: +, C. caerulens fatty acid synthetase; 0, yeast fatty acid synthetase; n , B. ammoniagenes fatty acid synthetase; A, yeast aicohol dehydrogenase. Preparations and assays of B. amwwniagenes and yeast fatty acid synthetases were carried out as described previously (7, 14). Assay of C. caerulens fatty acid synthetase was as described under Materials and Methods. Assay of yeast alcohol dehydrogenase (330 units/mg, Oriental Yeast Co. Ltd., Tokyo) was carried out according to the method of Racker (23).
34
KAWAGUCHI
the sensitivity of C. caerulens synthetase to cerulenin was much less than that of other synthetases. The enzymes from yeast and B. ammoniagenes were 50% inhibited by 1 pg/ml whereas C. caerulens synthetase was 50% inhibited by cerulenin at concentrations higher than 170 pg/ml. The activity of fatty acid synthetase from C. caerulens was not affected up to 10 pg/ml cerulenin and inhibition was not complete even at a concentration of 400 pg/ml. The inhibition at higher concentrations of cerulenin might be nonspecific because such high concentrations of cerulenin also caused the inhibition of yeast alcohol dehydrogenase to the same degree (Fig. 4). DISCUSSION
Antibiotic-producing organisms should have appropriate resistance to their own products. There have been several studies on the mechanisms of resistance that producing organisms display toward their own toxic metabolites and three different mechanisms were reported. InStreptomyces species which produce chloramphenicol, streptomycin, kanamycin, and neomycin, membrane permeability for these antibiotics plays an important role in the resistance of producers to these antibiotics (12,16,17). Permeability barriers prevent access of these antibiotics to the sites of protein synthesis which are the target of these antibiotics. These barriers presumably do not exist in cells grown in the absence of the antibiotics but develop after exposure to the antibiotics. In cerulenin-producing C. caerulens such development of a permeability barrier for cerulenin was not expected from the results in Fig. 3. However, from the results in Fig. 3 we can not rule out the possibility that C. caerulens has such a barrier per se. The second mechanism involving the resistance of the producers to their own antibiotics depends on inactivating enzymes of the antibiotics such as streptomycin kinase in S. grieseus (lg), neomycin kinase in S. fradiae (19)) kanamycin acetyltransferase in S. kanamyceticus (19, 20), and chloramphenicol hydrolase in Streptomyces species (21). We could not obtain data on the
ET AL.
presence of cerulenin-inactivating enzymes in C. caerulens. The third mechanism is the insensitivity of the target enzyme or a lack of the target site for the antibiotics. This is obvious in the case of penicillin-producing fungi which lack peptidoglycan, the target of their own antibiotic, in their cell walls. Antimycin Asensitive sites, which ought to lie between coenzyme Q or cytochrome b and cytochrome cl, are absent in a producer, S. antibioticus (22). In C. caerulens, the target enzyme, fatty acid synthetase, was found to be insensitive to cerulenin (Fig. 4). This fatty acid synthetase was purified to a homogenous state, judging from electrophoresis on agarose gel. Catalytic properties, the product patterns, and stereospecificities for reduced pyridine nucleotides of C. caerulens fatty acid synthetase were almost the same as those of yeast fatty acid synthetase. However, a striking difference between these fatty acid synthetases is their sensitivity to cerulenin. All fatty acid synthetases which have been isolated so far from cerulenin-sensitive organisms are very susceptible to cerulenin. Therefore, it is likely that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus. From this point of view, it seems of interest to determine the cerulenin-sensitivity of fatty acid synthetases from other species of the same genus, Cephalosporium, which are susceptible to cerulenin. D’Agnoloet al. (3) reported that cerulenin inhibition of p-ketoacyl-acyl carrier protein synthetase purified from Escherichia coli was irreversible and that it was accompanied by the binding of a single mole of cerulenin per mole of the enzyme. From this highly specific interaction between the enzyme and the inhibitor, they supposed that cerulenin inhibits the enzyme activity by alkylating the cystein residue at the active site. We also tried to determine the binding of cerulenin to the active site of the condensation reaction in fatty acid synthetases from C. caerulens and from B. ammoniagenes . Preliminary results showed that the amount of [3H]cerulenin bound to C. caerulens synthetase was about one-fifth
ISOLATION
OF CERULENIN-INSENSITIVE
of that bound to B. ammoniagenes synthetase. But it is apparent that a small amount of [3H]cerulenin was associated with C. caerulens synthetase. Judging from the chemical structure of cerulenin, the nucleophilic groups on a protein molecule (thiol, imidazole, etc.) presumably could attack the epoxide ring of cerulenin to form stable covalent bonding. It is certain that C. caerulens fatty acid synthetase carries a number of nucleophilic groups on its molecule. At the present stage, it is hard to dist,inguish the cerulenin binding to the active site of the condensation from that to other nucleophilic groups on the synthetase, since the condensing activity can not be isolated from multifunctional polypeptide chains of fatty acid synthetases. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Bloch, Department of Chemistry, Harvard University, for his interest and encouragement. We also thank Dr. Y. Seyama, Department of Biochemistry, Faculty of Medicine, The University of Tokyo, for the analysis of deuterated fatty acids by gc-ms and Dr. N. Ueta and Dr. Y. Miura, Department of Biochemistry, Teikyo University School of Medicine, for the analysis of radioactive fatty acids. The technical assistance of Miss N. Ugajin, School of Pharmaceutical Science, Kitasato University, is also gratefully acknowledged. This research was supported by grants-in-aid from the Ministry of Education of Japan. REFERENCES 1. OMURA, S., KATAGIRI, M., NAKAGAWA, A., SANO, Y., NOMURA, S., AND HATA, T. (1967)J. Antibiot. Ser. A 20, 349-354. 2. POUGNY, J. R., AND SINAY, P. (1978) Tetr. Lett. 1978, 3301-3304. 3. D’AGNOLO, G., ROSENFELD, I. S., AWAYA, J., OMURA, S., AND VAGELOS, P. R. (1973) Biochim. Biophys. Acta 326, 155-166.
FATTY
ACID
SYNTHETASE
35
4. VANCE, D., GOLDBERG, I., MITSUHASHI, O., BLOCH, K., OMURA, S., AND NOMURA, S. (1972) Bioehem. Biophys. Res. Commun. 48,649-656. 5. OMURA, S. (1976) Bacterial Rev. 40, 681-697. 6. IWAI, Y., AWAYA, J., KESADO, T., YAMADA, H., OMURA, S., AND HATA, T. (1973) J. Ferment. Technol. 51, 575-581. 7. KAWAGUCHI, A., ANDOKUDA, S. (1977)Proc. Nat. Acad. Sei. USA 74,3180-3183. 8. BRADFORD, M. M. (1976)Anal. Biochem. 72,248254. 9. NOMURA, S., HORIUCHI, T., OMURA, S., AND HATA, T. (1972) J. Biochem. 71, 783-796. 10. GOLDBERG, I., WALKER, J. R., AND BLOCH, K. (1973)Antimicrob. Ag. Chemother. 3,549-554. 11. DEES, C., AND OLIVER, J. D. (1977) Biochem. Rio-phys. Res. Commun. 78, 36-44. 12. MALIK, V. S., AND VINING, L. C. (1972) Canad. J. Microbial. 18, 583-590. 13. SEYAMA, Y., KASAMA, T., YAMAKAWA, T., KAWAGUCHI, A., AND OKUDA, S. (1977) J. Bio&em. 81, 1167-1173. 14. SEYAMA, Y., KASAMA, T., YAMAKAWA, T., KAWAGUCHI, A., SAITO, K., AND OKUDA, S. (1977) J. Biochem. 82, 1325- 1329. 15. SEYAMA, Y., KAWAGUCHI, A., KASAMA, T., SASAKI, K., ARAI, K., OKUDA, S., AND YAMAKAWA, T. (1978) Biomed. Mass Spectrom. 5, 357-361. 16. CELLA, R., AND VINING, L. C. (1974) Canad. J. Microbial. 20, 1591-1597. 17. HOT~A, K., AND OKAMI, Y. (1976) J. Ferment. Technol. 54, 563-571. 18. NIMI, O., ITO, G., SUEDA, S., ANDNOMI, R. (1971) Agr. Biol. Chem. 35, 848-858. 19. BENVENISTE, R., AND DAVIES, J. (1973) Proc. Nat. Acad. Sci. USA 70, 2276-2280. 20. SATOH, A., OGAWA, H., AND SATOMURA, Y. (1975) Agr. Biol. Chem. 39, 1593-1598. 21. MALIK, V. S., AND VINING, L. C. (1971) Canud. J. Microbial. 17, 1287-1290. 22. REHACEK, Z., RAMAKUTTY, M., AND KOZOVA, J. (1968) Appl. Microbial. 16, 29-32. 23. RACKER, E. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 506-503, Academic Press, New York.
AND BIOPHYSICS
Vol. 197, No. 1, October 1, pp. 30-35, 1979
Cerulenin
Resistance
in a Cerulenin-Producing
Fungus
Isolation of Cerulenin Insensitive Fatty Acid Synthetase AKIHIKO *institute
KAWAGUCHI,” HIROSHI TOMODA,* SHIGENOBU OKUDA,* JUICHI AWAYA,? AND SATOSHI &!IURAf
of Applied Microbiology, The University of Tokyo, Tokyo 113, and tThe Kitusuto School of Phm-maceutical Science, Kitasato University, Tokyo 108, Japan
Znstitute and
Received March 14, 1979; revised April 30, 1979 Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium cue&ens, is a potent inhibitor of fatty acid synthetase systems. This antibiotic specifically blocks the activity of P-ketoacyl thioester synthetase (condensing enzyme). The mechanism of the resistance of C. eaerulens to cerulenin was investigated. The rate of growth in medium containing up to 100 pg/ml cerulenin was as rapid as that in cerulenin-free medium. At a cerulenin concentration of 300 pg/ml, the rate of growth was still more than half that of the control. The addition of cerulenin (200 pg/ml) to a culture of growing cells has almost no effect on the incorporation of [‘%]acetate into cellular lipids. Fatty acid synthetase was purified from C. caerulens to homogeneity. Properties of this fatty acid synthetase were almost the same as those of yeast fatty acid synthetase except for the sensitivity to cerulenin. C. cue&ens synthetase is much less sensitive to cerulenin than fatty acid synthetases from other sources. These findings suggested that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus.
Cerulenin is an antifungal antibiotic isolated from a culture filtrate of the fungus Cephalosporium caerulens (1) and has the structure (2R)(3S)-2,3-epoxy-4-oxo-7,10dodecadienoylamide (2).l This antibiotic is a potent and noncompetitive inhibitor of fatty acid synthetase systems isolated from various microorganisms and from rat liver (3, 4). Low concentrations of cerulenin inhibit fatty acid synthetases of all known types, whether they are multienzyme complexes or nonaggregated systems. Further studies of the fatty acid synthetases purified from several organisms indicated that cerulenin specifically blocks the activity of P-ketoacyl thioester synthetase (condensing enzyme) (3, 4). Such a specificity has been useful for many investigators in studies of lipid metabolism and membrane biogenesis (5). We have investigated the mechanism of the resistance of C. caerulens to cerulenin.
We have already reported that the cerulenin producing strain, C. caerulens KF-140, was more resistant to cerulenin than the other species of the same genus, Cephalosporium (6). In this work, we purified fatty acid synthetase from C. caerulens and found that this synthetase was much less sensitive to cerulenin than fatty acid synthetases from other sources. This insensitivity of the enzyme to cerulenin may be responsible for the protection of C. caerulens from inhibitory effects of this antibiotic. MATERIALS
Organism and chemicals. Cephalosporium caerulens KF-140 was grown at 27°C with shaking in a medium containing 30 g of glycerol, 10 g of glucose, 5 g of peptone, and 2 g of NaCl per liter. Growth was recorded as the inerease in the absorbance at 660 nm. Cerulenin was prepared according to the method described previously (6). Sodium [2-%]acetate and [2-“C]malonyl-CoA were purchased from New England Nuclear, Boston; acetyl-CoA and malonyl-CoA were from P-L Biochemicals Inc., Milwaukee, Wiscon-
1 The absolute configuration of epoxide is (2R)(3S) instead of (2S)(3R) as previously reported (1). 0003-9361/79/110030-06$02.00/o
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
30
ISOLATION
OF CERULENIN-INSENSITIVE
FATTY ACID SYNTHETASE
31
sin; NADPH and NADH were from Oriental Yeast Co., Tokyo. All other reagents and solvents were of analytical grade. Microbial assay of cerulenin. The amount of cerulenin was determined by microbial assay employing Candida albicans KF-1 as the test microorganism according to the method reported previously (6). Incorporation of [%‘]acetate into lipids in vivo. C. cue&ens cells, which were grown at 27’C for 6, 24,
and 48 h, respectively, were centrifuged, and resuspended in the growth medium. The turbidity of each cell suspension was adjusted to 0.2 of the absorbance at 660 nm. Aqueous cerulenin solution (0.1 ml) was added to 0.8-ml portions of cell suspensions and the mixtures were incubated at 27°C for 10 min. Subsequently, 0.1 ml of [Wlacetate (0.2 &i) was added to each mixture and incubation was continued for a further 15 min. One milliliter of 10% trichloroacetic acid was added and the cells were washed twice with 5% trichloroacetic acid. Lipids were extracted from the cells with chloroform-methanol (2:1, v/v). Samples of the chloroform-methanol extracts were evaporated to dryness and the radioactivity counted with a lipid scintillation counter. Preparation
of cell-free extracts from C. cue&ens.
Ten grams of C. caerulens cells was suspended in 20 ml of 0.1 M potassium phosphate buffer (pH 7.0) and disrupted for 2 min with 15 g of glass beads in a Braun homogenizer. Cell debris was removed by centrifugation at 10,OOOgfor 5 min and the supernatant was centrifuged again at 80,OOOgfor 1 h. The resulting supernatants were used as the cell-free extracts. Fatty acid synthetase assay. Fatty acid synthetase activity was assayed spectrophotometrically according to the method described previously (7). Unless otherwise stated, assays were carried out at 37°C in mixtures containing 0.1 M potassium phosphate buffer (pH 7.15), 5 mM dithiothreitol, 200 pM NADPH, 50 pM acetyl-CoA, 40 pM malonyl-CoA, 100 pg bovine serum albumin, and enzyme protein in a total volume of 0.5 ml. One unit of activity is defined as the amount of enzyme required to incorporate 1 nmol of malonyl-CoA per minute into fatty acids. Purification offatty acid synthetase. All steps were performed at 0-4°C. Protein was determined by the
FIG. 1. Agarose gel electrophoresis of C. caerulens fatty acid synthetase. Gel electrophoresis on 1% agarose was done according to the method previously described (7). After the gels were destained, they were scanned with a Fujiox (Tokyo, Japan) densitometer. method of Bradford (8). All buffers contained 10 mM 2-mercaptoethanol and 1 mM EDTA, unless otherwise stated. For the preparation of fatty acid synthetase, C. cue&ens was grown to the mid-log phase, for 24 h. Fifty-four grams of frozen cells was thawed in 216 ml of 0.1 M potassium phosphate buffer (pH 7.0). The cells were broken by passage through a French pressure cell operated at 20,000 psi and the disrupted cells were centrifuged at 25,000g for 20 min. The protein concentration of the resulting supernatant was adjusted to 10 mg/ml by adding 0.1 M potassium phosphate buffer (pH 7.0) and the diluted supernatant was brought to 30% saturation with ammonium sulfate. After stirring for 15 min, the precipitate was removed by centrifugation at 25,000g for 20 min and discarded. The supernatant was brought to 50% saturation with ammonium sulfate, stirred for 15 min, and centrifuged at 25,000g for 20 min. The precipitate was dissolved in 18 ml of 0.05 M potassium phosphate buffer (pH 7.0) and dialyzed against 0.05 M potassium phosphate buffer (pH 7.0) for 3 h. The dialyzed solution was applied to a DEAE-cellulose column (2.4 x 20 cm) that had previously been equilibrated with 0.05 M potassium phosphate buffer (pH 7.0). The enzyme was eluted by a linear gradient from 0.05 to 0.25 M potassium phosphate, pH 7.0 (600 ml). The major fractions containing
TABLE I PURIFICATIONOF FATTY ACID SYNTHETASEFROMC. cue&ens
Fraction
Volume (ml)
Crude extract (NH&SOI, 30-50% DEAE-cellulose Sepharose 6B
280 18 120 8.5
Total activity (unit) 29300 11600 7350 1620
Total protein (mg)
Specific activity (unit/mg)
Yield (%)
6020 1190 58.9 3.0
4.87 9.75 125 541
100 39.5 25.0 5.5
32
XAWAGUCHI
enzyme activity were brought to 50% saturation with ammonium sulfate and centrifuged at 25,000g for 20 min. The precipitate was dissolved in 2 ml of 0.25 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1 mM dithiothreitol instead of 10 mM 2mercaptoethanol, and applied to a Sepharose 6B column (1.7 x 50 cm) that had been equilibrated with the same buffer. The fractions containing enzyme activity were combined and concentrated with a Diaflo apparatus with an XM-50 membrane. The results of this purification are summarized in Table I. After the final purification step the synthetase was homogenous, as judged by the appearance of a single band when subjected to electrophoresis on 1% agarose gels (Fig. 1). RESULTS
Effect of Cerulenin on the Growth of C. caerulens A previous paper from our laboratory reported that the growth of C. caerulens reached the stationary phase after about 50 h of cultivation (6). Cerulenin was detected in the culture filtrate after 12 h of cultivation and the amount of cerulenin reached a maximum (about 250 pg/ml) at 48-50 h. To reveal the cerulenin resistance of C. caerulens, growth of this fungus in medium supplemented with cerulenin was carried out and the results are shown in Fig. 2. Early (Fig. 2A) and late (Fig. 2B) logphase cells were transferred to the medium supplemented with cerulenin and the growth was followed by measuring the absorbance at 660 nm. Both the length of the lag phase and the rate of the growth were slightly affected by the age of the inoculum but the
ET AL.
0
100 CERULENIN (pglml )
200
FIG. 3. Cerulenin effect on the incorporation of [14C]acetate into the lipid fraction of C. caerulens. See Materials and Methods for details. Cells grown for 6 (+I, 24 (O), and 48 h (A) were used for experiments. Results are expressed as percentages of 14Cincorporation relative to experiments in which cell suspensions were treated without cerulenin.
growth patterns were essentially the same. The growth rate in the medium containing up to 100 pg/ml cerulenin was as rapid as that in cerulenin-free medium and even with 300 pg/ml cerulenin the growth rate was more than half the rate in the controls. Since the growth of cerulenin sensitive organisms was stopped completely at cerulenin concentration of 100 pg/ml (5), the above results suggested that C. caerulens exhibited a high resistance to cerulenin. Malik and Vining (12) studied the chloramphenicol resistance of chloramphenicolproducing Streptomyces and observed that late log-phase cells showed higher resistance to chloramphenicol than early logphase cells. To explain these observations, they postulated that membrane permeability for the antibiotic changed after the exposure to chloramphenicol. In the case of C. caerulens, it is hard to consider such changes of membrane permeability after exposure to cerulenin, because the growth patterns were not dependent on the age of the inoculum. Effect of Cerulenin on Lipid Synthesis
FIG. 2. Cerulenin effect on the growth of C. caerulens. Cells grown for 8 (A) and 48 h (B) were inoculated into L-tubes, each containing 5 ml of medium supplemented with various concentrations of cerulenin (numbers in parentheses indicate pg/ml). See Materials and Methods for details.
The effect of cerulenin on the incorporation of [14C]acetate into cellular lipids was studied with a growing of C. caerulens (Fig. 3). Growing cultures of three different phases were transferred to fresh medium
ISOLATION
OF CERULENIN-INSENSITIVE
containing [14C]acetate and cerulenin, and the incorporation of acetate into the lipid fractions was measured. Many investigators have confirmed that cerulenin specifically interferes with the incorporation of acetate into lipids in a variety of yeasts, fungi, and bacteria (9-11). In these cerulenin sensitive organisms, 100 pg/ml cerulenin completely inhibits the incorporation of labeled precursor into lipid fractions. On the contrary, lipid synthesis in C. caerulens was inhibited only slightly by cerulenin. The enzyme system for lipid synthesis was still active even at a concentration of 200 @g/ml cerulenin. These results were also confirmed by the experiments with cell-free extracts of C. caerulens. Cell-free extracts prepared from C. caerwlens cells of both mid- and late log phases retained the activity of fatty acid synthesis from [2-14C]malonyl-CoA up to 400 pg/ml cerulenin. Cerulenin was found to retain almost full antibiotic activity during a 30-min incubation with growing cell suspensions and with cell-free extracts. From these results we can rule out the possibility that cerulenin was inactivated during the above reaction of fatty acid synthesis. Since the principal target of cerulenin has been established to be fatty acid synthesis, it is reasonable to consider that the fatty acid synthetase system of this fungus is resistance to cerulenin. Putification and Properties Synthetase
FATTY
ACID
33
SYNTHETASE
for reduced pyridine nucleotide(s). It was found that the stereospecificity of enoyl reductase was dependent on the source of the enzyme; rat liver enzyme was A-specific and enzymes from Brevibacterium ammoniagenes and yeast were B-specific (13-15). The stereospecificity of the fatty acid synthetase from C. caerulens was also determined by the same technique as reported previously (13). Both /3-ketoacyl reductase and enoyl reductase were B-specific. The properties of C. caerulens fatty acid synthetase are similar to those of yeast fatty acid synthetase. Inhibition
of Fatty Acid Synthetases
by
Cerulenin Figure 4 shows the effects of cerulenin on fatty acid synthetases purified from yeast, B. ammoniagenes and C. caerulens, and also on alcohol dehydrogenase from yeast. Cerulenin inhibition of yeast fatty acid synthetase was complete with 5 pglml. Fatty acid synthetase of B. ammoniagenes was as sensitive to cerulenin as yeast synthetase. Vance et al. (4) also reported that fatty acid synthetases of Euglena gracilis, Corynebacterium diphutheriae, and Mycobacterium phlei were inhibited by cerulenin to almost the same extent. On the contrary,
of Fatty Acid
To confirm the conclusion that fatty acid synthetase itself is resistant to cerulenin, we purified fatty acid synthetase from C. caerulens (Table I) and obtained a homogeneous preparation of this enzyme (Fig. 1). The pH optimum of the enzyme activity was 7.15. The apparent K, values for the three substrates were: Malonyl-CoA, 9.1 PM; acetyl-CoA, 9.3 PM; and NADPH, 14 PM. The molecular weight, estimated by chromatography on Sepharose 6B, was estimated to be 1.2 x lo6 in three trials. Under the standard assay conditions, 41% of the fatty acids produced is palmitate and 59% stearate. Recently we determined the stereospecificities of P-ketoacyl and enoyl reductases (partial reactions of fatty acid synthetase)
” 10
50
100
200 CERULENIN
300
400
(pglml)
FIG. 4. Percentage inhibition of fatty acid synthetases and yeast alcohol dehydrogenase by cerulenin. The symbols in the graph are: +, C. caerulens fatty acid synthetase; 0, yeast fatty acid synthetase; n , B. ammoniagenes fatty acid synthetase; A, yeast aicohol dehydrogenase. Preparations and assays of B. amwwniagenes and yeast fatty acid synthetases were carried out as described previously (7, 14). Assay of C. caerulens fatty acid synthetase was as described under Materials and Methods. Assay of yeast alcohol dehydrogenase (330 units/mg, Oriental Yeast Co. Ltd., Tokyo) was carried out according to the method of Racker (23).
34
KAWAGUCHI
the sensitivity of C. caerulens synthetase to cerulenin was much less than that of other synthetases. The enzymes from yeast and B. ammoniagenes were 50% inhibited by 1 pg/ml whereas C. caerulens synthetase was 50% inhibited by cerulenin at concentrations higher than 170 pg/ml. The activity of fatty acid synthetase from C. caerulens was not affected up to 10 pg/ml cerulenin and inhibition was not complete even at a concentration of 400 pg/ml. The inhibition at higher concentrations of cerulenin might be nonspecific because such high concentrations of cerulenin also caused the inhibition of yeast alcohol dehydrogenase to the same degree (Fig. 4). DISCUSSION
Antibiotic-producing organisms should have appropriate resistance to their own products. There have been several studies on the mechanisms of resistance that producing organisms display toward their own toxic metabolites and three different mechanisms were reported. InStreptomyces species which produce chloramphenicol, streptomycin, kanamycin, and neomycin, membrane permeability for these antibiotics plays an important role in the resistance of producers to these antibiotics (12,16,17). Permeability barriers prevent access of these antibiotics to the sites of protein synthesis which are the target of these antibiotics. These barriers presumably do not exist in cells grown in the absence of the antibiotics but develop after exposure to the antibiotics. In cerulenin-producing C. caerulens such development of a permeability barrier for cerulenin was not expected from the results in Fig. 3. However, from the results in Fig. 3 we can not rule out the possibility that C. caerulens has such a barrier per se. The second mechanism involving the resistance of the producers to their own antibiotics depends on inactivating enzymes of the antibiotics such as streptomycin kinase in S. grieseus (lg), neomycin kinase in S. fradiae (19)) kanamycin acetyltransferase in S. kanamyceticus (19, 20), and chloramphenicol hydrolase in Streptomyces species (21). We could not obtain data on the
ET AL.
presence of cerulenin-inactivating enzymes in C. caerulens. The third mechanism is the insensitivity of the target enzyme or a lack of the target site for the antibiotics. This is obvious in the case of penicillin-producing fungi which lack peptidoglycan, the target of their own antibiotic, in their cell walls. Antimycin Asensitive sites, which ought to lie between coenzyme Q or cytochrome b and cytochrome cl, are absent in a producer, S. antibioticus (22). In C. caerulens, the target enzyme, fatty acid synthetase, was found to be insensitive to cerulenin (Fig. 4). This fatty acid synthetase was purified to a homogenous state, judging from electrophoresis on agarose gel. Catalytic properties, the product patterns, and stereospecificities for reduced pyridine nucleotides of C. caerulens fatty acid synthetase were almost the same as those of yeast fatty acid synthetase. However, a striking difference between these fatty acid synthetases is their sensitivity to cerulenin. All fatty acid synthetases which have been isolated so far from cerulenin-sensitive organisms are very susceptible to cerulenin. Therefore, it is likely that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus. From this point of view, it seems of interest to determine the cerulenin-sensitivity of fatty acid synthetases from other species of the same genus, Cephalosporium, which are susceptible to cerulenin. D’Agnoloet al. (3) reported that cerulenin inhibition of p-ketoacyl-acyl carrier protein synthetase purified from Escherichia coli was irreversible and that it was accompanied by the binding of a single mole of cerulenin per mole of the enzyme. From this highly specific interaction between the enzyme and the inhibitor, they supposed that cerulenin inhibits the enzyme activity by alkylating the cystein residue at the active site. We also tried to determine the binding of cerulenin to the active site of the condensation reaction in fatty acid synthetases from C. caerulens and from B. ammoniagenes . Preliminary results showed that the amount of [3H]cerulenin bound to C. caerulens synthetase was about one-fifth
ISOLATION
OF CERULENIN-INSENSITIVE
of that bound to B. ammoniagenes synthetase. But it is apparent that a small amount of [3H]cerulenin was associated with C. caerulens synthetase. Judging from the chemical structure of cerulenin, the nucleophilic groups on a protein molecule (thiol, imidazole, etc.) presumably could attack the epoxide ring of cerulenin to form stable covalent bonding. It is certain that C. caerulens fatty acid synthetase carries a number of nucleophilic groups on its molecule. At the present stage, it is hard to dist,inguish the cerulenin binding to the active site of the condensation from that to other nucleophilic groups on the synthetase, since the condensing activity can not be isolated from multifunctional polypeptide chains of fatty acid synthetases. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Bloch, Department of Chemistry, Harvard University, for his interest and encouragement. We also thank Dr. Y. Seyama, Department of Biochemistry, Faculty of Medicine, The University of Tokyo, for the analysis of deuterated fatty acids by gc-ms and Dr. N. Ueta and Dr. Y. Miura, Department of Biochemistry, Teikyo University School of Medicine, for the analysis of radioactive fatty acids. The technical assistance of Miss N. Ugajin, School of Pharmaceutical Science, Kitasato University, is also gratefully acknowledged. This research was supported by grants-in-aid from the Ministry of Education of Japan. REFERENCES 1. OMURA, S., KATAGIRI, M., NAKAGAWA, A., SANO, Y., NOMURA, S., AND HATA, T. (1967)J. Antibiot. Ser. A 20, 349-354. 2. POUGNY, J. R., AND SINAY, P. (1978) Tetr. Lett. 1978, 3301-3304. 3. D’AGNOLO, G., ROSENFELD, I. S., AWAYA, J., OMURA, S., AND VAGELOS, P. R. (1973) Biochim. Biophys. Acta 326, 155-166.
FATTY
ACID
SYNTHETASE
35
4. VANCE, D., GOLDBERG, I., MITSUHASHI, O., BLOCH, K., OMURA, S., AND NOMURA, S. (1972) Bioehem. Biophys. Res. Commun. 48,649-656. 5. OMURA, S. (1976) Bacterial Rev. 40, 681-697. 6. IWAI, Y., AWAYA, J., KESADO, T., YAMADA, H., OMURA, S., AND HATA, T. (1973) J. Ferment. Technol. 51, 575-581. 7. KAWAGUCHI, A., ANDOKUDA, S. (1977)Proc. Nat. Acad. Sei. USA 74,3180-3183. 8. BRADFORD, M. M. (1976)Anal. Biochem. 72,248254. 9. NOMURA, S., HORIUCHI, T., OMURA, S., AND HATA, T. (1972) J. Biochem. 71, 783-796. 10. GOLDBERG, I., WALKER, J. R., AND BLOCH, K. (1973)Antimicrob. Ag. Chemother. 3,549-554. 11. DEES, C., AND OLIVER, J. D. (1977) Biochem. Rio-phys. Res. Commun. 78, 36-44. 12. MALIK, V. S., AND VINING, L. C. (1972) Canad. J. Microbial. 18, 583-590. 13. SEYAMA, Y., KASAMA, T., YAMAKAWA, T., KAWAGUCHI, A., AND OKUDA, S. (1977) J. Bio&em. 81, 1167-1173. 14. SEYAMA, Y., KASAMA, T., YAMAKAWA, T., KAWAGUCHI, A., SAITO, K., AND OKUDA, S. (1977) J. Biochem. 82, 1325- 1329. 15. SEYAMA, Y., KAWAGUCHI, A., KASAMA, T., SASAKI, K., ARAI, K., OKUDA, S., AND YAMAKAWA, T. (1978) Biomed. Mass Spectrom. 5, 357-361. 16. CELLA, R., AND VINING, L. C. (1974) Canad. J. Microbial. 20, 1591-1597. 17. HOT~A, K., AND OKAMI, Y. (1976) J. Ferment. Technol. 54, 563-571. 18. NIMI, O., ITO, G., SUEDA, S., ANDNOMI, R. (1971) Agr. Biol. Chem. 35, 848-858. 19. BENVENISTE, R., AND DAVIES, J. (1973) Proc. Nat. Acad. Sci. USA 70, 2276-2280. 20. SATOH, A., OGAWA, H., AND SATOMURA, Y. (1975) Agr. Biol. Chem. 39, 1593-1598. 21. MALIK, V. S., AND VINING, L. C. (1971) Canud. J. Microbial. 17, 1287-1290. 22. REHACEK, Z., RAMAKUTTY, M., AND KOZOVA, J. (1968) Appl. Microbial. 16, 29-32. 23. RACKER, E. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 506-503, Academic Press, New York.