J. Biochem. 85, 1531-1537 (1979)

Studies on J8-zF Isomerization and Methyl Transfer of Sterols in Ergosterol Biosynthesis of Yeast1 Yoshiyasu YABUSAKI, 1 Tokuzo NISHINO, Nakao ARIGA 3 , and Hirohiko KATSUKI 4 Department of Chemistry, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, January 29, 1979

The formation of cholesta-7,24-dien-3£-ol and its activity as a substrate for the sterol sidechain methyltransferase in yeast have not previously been studied. Experiments with acetonepowder extracts of yeast showed that the sterol is formed from zymosterol by J 8 -J 7 isomerization. However, direct conversion of cholesta-7,24-dien-3j9-ol into zymosterol could not be demonstrated. The reversibility of the reaction was proved by the detection of *H-incorporation into cholesta-8-en-3/9-ol (with lathosterol as a carrier) from [3H]H,O in the medium. Incubation of cholesta-7,24-dien-3£-ol and 5-adenosyl-L-{methyl-14C]methionine with the acetone-powder extract resulted in methylation of the sterol to form episterol. Similar incubation of zymosterol gave fecosterol and episterol, suggesting that fecosterol initially formed by the methylation was isomerized to episterol. In intact cells, however, an alternative pathway (zymosterol —> cholesta-7,24-dien-3/3-ol —»episterol) may also operate. The relative importance of the two pathways is not known.

Two theories have been proposed regarding the introduction of 28-Me during ergosterol biosynthesis in yeast. Barton et al. claimed that the methylation occurs before complete elimination 1

This investigation was supported in part by research grants from the Ministry of Education, Science and Culture of Japan, and Ajinomoto Co., Inc. 1 Present address: Research Department, Pesticides Division, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665. ' Present address: Department of Chemistry, Faculty of General Education, Gifu University, Nagara, Gifu 502. 4 To whom correspondence should be addressed. Abbreviations: GSH, glutathionine; DTT, dithiothreitol; J 7 ' M -C,,, cholesta-7,24-dien-30-ol. Vol. 85, No. 6, 1979

of the three methyl groups attached to the sterol nucleus on the basis of sterol feeding experiments with intact cells (1, 2). In contrast, Katsuki and Bloch concluded that the methylation occurs at the stage of C,,-sterol(s), based on the observation that all of the methylated sterols were ergostane derivatives in a cell-free reaction system (J). Moore and Gaylor subsequently solubilized and purified 5-adenosylmethionine: JM-sterol methyltransferase from yeast (4) and reported that zymosterol was the best substrate among the sterols tested, while sterols with an extra methyl group(s) on the nucleus had very little or no activity as substrates (5). Our recent paper reporting the accumulation of zymosterol in cells grown in the presence of 1531

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Y. YABUSAKI, T. NISHINO, N. ARIGA, and H. KATSUKJ

ethionine to prevent the methylation of sterols seemed to support the view that the methyl acceptor was zymosterol (6). In experiments with cell-free extracts by our group, however, J 7 - M -C, 7 was found to accumulate when the methylation was inhibited by homocysteine (7). This was reported by three other groups at almost the same time (8-10). Recently Osumi et al. reported multiple pathways in ergosterol biosynthesis, including a pathway through ^ 7 - M -C, 7 (11). On the basis of our (11) and other (4, 5) results, Q.7-sterol(s) plays a major role and 4-methyl zymosterol a minor role in methylation in yeast. However, details of the enzymic formation and methylation of J 7>M -Ci 7 have not yet been reported. J 7 - M -C, 7 was demonstrated to be formed enzymically by J 8 -A 1 isomerization from zymosterol in animals (12, 13). Although the reactions in sterol synthesis of yeast are similar to those of animals in many respects, different mechanisms have been proposed for some reactions. It was reported that the 7 /3-hydrogen atom was eliminated in J 8 -J 7 isomerization in rat liver, but the 7ahydrogen atom in yeast (14, 15). The present paper describes studies on J 8 -J 7 isomerization and some of the properties of the enzyme involved, and also deals with the methylation of ^ 7>M -Cn in yeast. MATERIALS AND METHODS Chemicals—S- Adenosyl - L - [methyl - U C] methi onine (59 Ci/mol) was purchased from the Radiochemical Centre, Amersham. Sodium PHjborohydride and PH]H,O were kindly provided by Prof. E. Fujita and J. Hayami of Kyoto University, respectively. Desmosterol was kindly provided by Prof. M. Dcekawa and Dr. M. Morisaki of Tokyo Institute of Technology. Lanosterol, GSH, DTT, NADH, and NADPH were obtained from Sigma and lathosterol from Schwarz/Mann. Kieselguhr G (Art. 8129) and thin-layer chromatographic plates (Silica Gel) were from Merck. All other chemicals were of analytical grade. Growth of Yeast and Preparation of Cell-Free Extracts—Saccharomyces cerevisiae ATCC 12341 was grown as described by Katsuki and Bloch (5) and was disrupted with a Vibrogen cell mill (Edmond Muhler) in 0.1 M potassium phosphate

buffer (pH 6.8) containing 0.1 HIM DTT. The homogenate was centrifuged at 2,500 xg for 10 min and the resulting supernatant was used as cell-free extract. Preparation of acetone powder of the cell-free extract was carried out as described by Moore and Gaylor (4). The acetone powder was suspended in 0.1 M potassium phosphate buffer (pH 6.8) and the resulting extract was used as the enzyme. Preparation of Sterols for Use as Substrates— Zymosterol was prepared according to the method of Ariga et al. (6) as follows: the yeast was grown in the presence of ethionine and the sterols were extracted with petroleum ether after saponification treatment of the cells. Separation of zymosterol from other sterols was carried out by thin-layer chromatography on Kieselguhr G using cyclohexane-ethyl acetate (99.5 :0.5, v/v) as a solvent. Fecosterol and episterol were prepared as follows: the cells were heated with alkali for saponification and non-saponifiable lipids were extracted with petroleum ether. They were acetylated and separated by thin-layer chromatography on Silica Gel G impregnated with 20% AgNO, according to the method of Fryberg et al. (16). Benzene was used as the developing solvent. The sterol acetates were visualized under UV light by spraying 0.2% 2',7'-dicnlorofluorescein dissolved in ethanol, then the sterol acetates were scraped from the plate and extracted with nhexane. The purity of these sterol acetates was checked by gas-liquid chromatography using a 1.5% OV-17 column. Alkali hydrolysis yielded the corresponding free sterols. The preparation of J7iM-Ct7 will be described in a subsequent section. Synthesis of [*H] Zymosterol—Zymosterol was oxidized with tert-butyl chromate reagent in CC14 by the method of Menini and Norymberski (17). The oxidized compound, zymosterone (about 1 mg), was reduced with sodium boro[*H]hydride (about 100 /iCi) in methanol. The resulting fH]zymosterol, a mixture of 3a- and 3/9-hydroxylated isomers, was subjected to thin-layer chromatography by the method of Anding et al. (18). The /3-isomer (Rf, 0.40) was separated from the aisomer (Rf, 0.55) and used for the enzyme reaction. Assays of Enzymes—As-A1 Isomerase: The standard assay mixture contained the following constituents in a final volume of 1 ml: 40 //mol of / . Biochem.

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J'-d 7 ISOMERJZATION AND METHYLATION OF STEROLS IN YEAST zymosterol, 10 /imol of DL-homocysteine, of potassium phosphate buffer (pH 6.8), and the enzyme (10 mg of protein). Tween 80 at a final concentration of 0.2% was used to disperse the substrate. It was incubated for 1.5-2 h at 30°C with shaking. The reaction was stopped by the addition of 1 g of KOH and 3 ml of methanol, and the mixture was saponified. Non-saponifiable lipids were extracted with petroleum ether. The activity of isomerase was assayed by the determination of J 7 '"-Ci r formed by gas-liquid chromatography. For the preparation of J 7 ' ! 4 - Q 7 , the enzyme reaction was carried out on a larger scale. Methyltransferase: The standard assay mixture contained the following constituents in a final volume of 1 ml: 5-adenosyl-L-[methyl-14C]methionine (0.05 pCi), 15.6 /imol of GSH, 10 /imol of MgCl,, 40 /imol of sterol as a substrate, 100 /imol of potassium phosphate buffer (pH 6.8), and the enzyme (10 mg of protein). The method for dispersion of sterols was as described above. Incubation was carried out for 30 min at 30°C with shaking. The activity of methyltransferase was determined by measuring the radioactivity incorporated from iSr-adenosyI-L-[methyI-14C]methionine into sterols using a Beckmann LS-230 liquid scintillation spectrometer, with toluene scintillator. Analysis of Sterols—Analysis of radioactive sterols was carried out with a Shimadzu GC-5A gas chromatograph equipped with a Shimadzu RID-2E radioisotope detector after conversion to methyl-ethers or trimethylsilyl derivatives. A glass column (4 mm i.d. and 1 m long) packed with 5 % diethyleneglycol succinate adsorbed on Chromosorb W (80-100 mesh) or 10% Silar 10C adsorbed on Gas Chrom Q (100-120 mesh) was used. The column temperature was 190°C. For the analysis of s H-labeled sterols, the substances emerging from the gas chromatograph every 1 min were trapped in a glass U-tube cooled in ice and the radioactivities were measured (the recovery of radioactive sterol was about 85 %). When nonradioactive zymosterol was used as a substrate, a Shimadzu GC-6AM gas chromatograph equipped with a flame ionization detector was used. The column used was a SCOT glass capillary column (0.3 mm i.d. and 50 m long) coated with OV-17 supported on Silanox, and the flow rate of carrier

1533

gas (Nt) was 1 ml/min with a split ratio of 1 : 31 (temperature, 250 c C). Cholesterol was used as an internal standard. Determination of Protein—Protein concentration was determined by the method of Lowry et al. (19). RESULTS

Characterization of the J 8 -J 7 Isomerization Reaction with Cell-Free Extract—[*H]Zymosterol was incubated with the cell-free extract for 3 h at 30°C under anaerobic conditions in the presence of DL-homocysteine and DL-ethionine, which were added to prevent sterol side-chain methylation. After the reaction, non-saponifiable lipids were extracted and converted to methyl-ether derivatives for determination by gas-liquid chromatography (Fig. 1). The chromatogram showed a new peak with a larger retention time than that of zymosterol. The retention time of the product relative to that of zymosterol accorded with the literature value for J 7 ' M -C, 7 (20). For confirmation, the mass spectrum of the trimethylsilyl derivative of the product was taken (m\e 343 (M-side-chain-2H), 255 (M-90-side-chain), and 213 (M-90-side-chain42)); it was in accord with that reported by Parks et al. (8). Accordingly, the compound was identified as J 7 '"-C,,, indicating that it was formed by isomerization of zymosterol. Properties of J 8 -J 7 Isomerase—The acetonepowder extract contained both the methyltransferase and the J 8 -J' isomerase, but attempts to separate them were unsuccessful. However, since the extract contained no endogenous sterols, they were used as the enzyme throughout. In further experiments, non-labeled zymosterol could be used as a substrate and the enzyme activity was assayed by determination of the amount of /J7'"-C,7 produced by gas-liquid chromatography. Table I shows requirements for oxygen and cofactors for the enzyme reaction. The enzyme reaction proceeded under anaerobic as well as aerobic conditions. About the same enzyme activity was seen in the absence of NADH, NADPH, GSH, M g ' \ and Mn1+ as in their presence. These results indicate that the isomerase reaction in yeast does not require oxygen and cofactors, as is the case in animals. The enzyme reaction proceeded linearly for at

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Y. YABUSAKJ, T. NISHINO, N. ARJGA, and H. KATSUKJ

1534 A. Zymo

3 2

I Retention Time ( mln )

CO

B. O15 ^
M-C,7 from zymosterol appears to be less than 10% under the experimental conditions used, based on the kinetic properties of the isomerase; ii) the rate of isomerization from fecosterol to episterol is faster than that from zymosterol to -47'"-Ct,; iii) the methylation activities towards zymosterol and /T' M -Cj 7 are about the same. In the intact cells, in contrast, it is possible that both pathways, (a) and (b), operate. However, it is difficult to determine which is the main route and whether or not they operate together. The presence of sterols in free and esterified forms in yeast complicates the situation. Moreover, sterol esters are known not to serve as substrates for isomerization and methylation. Recent investigations in our laboratory on the substrate specificity of sterol-ester synthetase showed that most of the sterols tested could serve as substrates to about the same extent (23). However, it is known that most of the zymosterol is present in an esterified form, whereas a considerable amount of J 7 '"-C, 7 is present in a free form in yeast (24). If it is considered that the equilibrium of the J s -/J 7 isomerization reaction lies far to the 47-sterol side, the fact that the amount of zymosterol is far greater than that of J 7 -"-C f7 in yeast cells is difficult to explain. Some control mechanism must be involved in the isomerization, methylation, and esterification of sterols. The authors wish to express their gratitude to Profs. S. Seto and K. Ogura of Tohoku University for GC-MS measurements. Thanks arc due to Profs. E. Fujita and J. Hayami of Kyoto University for the kind gift of tritium compounds. Thanks are also due to Prof. M. Dcekawa and Dr. M. Morisaki of Tokyo Institute of Technology for the kind gift of desmosterol. We also thank the Central Research Laboratories, Ajinomoto Co., Inc. for financial support.

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A>-/T ISOMERIZATION AND METHYLATION OF STEROLS IN YEAST

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11.

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12. Dempsey, M.E. (1965) /. Biol. Chem. 240,4176-4188 13. Yamaga, N. & Gaylor, J.L. (1978) / . Lipid. Res. REFERENCES 19, 375-382 Barton, D.H.R., Harrison, D.M., & Widdowson, 14. Caspi, E. & Ramm, P.J. (1969) Tetrahedron Lett. 181-185 D.A. (1968) Chem. Commun. 17-19 Barton, D.H.R., Harrison, D.M., Moss, G.P., & 15. Akhtar, M., Rahimtula, A.D., & Wilton, D.C. (1970) Biochem. J. 117, 539-542 Widdowson, D.A. (1970) / . Chem. Soc. 775-785 Katsuki, H. & Bloch, K. (1967) / . Biol. Chem. 244, 16. Fryberg, M., Oehlschlager, A.C., & Unrau, A.M. (1973) / . Am. Chem. Soc. 95, 5747-5757 222-227 Moore, J.T., Jr. & Gaylor, J.L. (1969) /. Biol. 17. Menini, E. & Norymberski, J.K. (1962) Biochem. J. 84, 195-201 Chem. 244, 6334-6340 Moore, J.T., Jr. & Gaylor, J.L. (1970) /. Biol. 18. Anding, C , Parks, L.W., & Ourisson, G. (1974) Eur. J. Biochem. 43, 459-463 Chem. 245, 4684-4688 Ariga, N., Hatanaka, H., Nagai, J., & Katsuki, H. 19. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randal], R.J. (1951) / . Biol. Chem. 193, 265-275 (1978) /. Biochem. 83, 1109-1116 Hatanaka, H., Ariga, N., Nagai, J., & Katsuki, H. 20. Clayton, R.B. (1962) Biochemistry 1, 357-366 (1974) Biochem. Biophys. Res. Commun. 60, 787-793 21. Gaylor, J.L., Delwiche, C.V., & Swindell, A.C. (1966) Steroids 8, 353-362 Parks, L.W., Anding, C , & Ourisson, G. (1974) 22. Wilton, D.C, Rahimtula, A.D., & Akhtar, M. Eur. J. Biochem. 43, 451^*58 (1969) Biochem. J. 114, 71-73 Barton, D.H.R., Gunatilaka, A.A.L., Jarman, T.R., Widdowson, D.A., Bard, M., & Woods, R.A. 23. Taketani, S., Nishino, T., & Katsuki, H. Biochim. Biophys. Ada in press (1975) / . Chem. Soc. 88-92 Morcau, J.P., Ramra, P.J., & Caspi, E. (1975) 24. Taketani, S., Nagai, J., & Katsuki, H. (1978) Biochim. Biophys. Ada 528, 416-423 Eur. J. Biochem. 56, 393-402 Osumi, T., Taketani, S., Katsuki, H., Kuhara, T., & Matsumoto, I. (1978) / . Biochem. 83, 681-691

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Studies on delta8-delta7 isomerization and methyl transfer of sterols in ergosterol biosynthesis of yeast.

J. Biochem. 85, 1531-1537 (1979) Studies on J8-zF Isomerization and Methyl Transfer of Sterols in Ergosterol Biosynthesis of Yeast1 Yoshiyasu YABUSAK...
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