Δ14-sterol reductase in Saccharomyces cerevisiae

301

Biochimica et Biophysics Actu, 531 (1978) 301-307 0 Eisevier/North-H~liand Biomedical Press

BBA 57279

A’4-STEROLREDUCTASEIN

SACCIiAROMYCESCEREVZSZAE

C.K. BOTTEMA and L.W. PARKS * Department

of Microbiology,

Oregon State

University,

Corvallis, Oreg. 97331

(U.S.A.)

(Received June 12th, 1978)

Summary An in vitro assay for A14-sterol reductase from yeast was developed, using ergosta-8,14dien-S&o1 as the substrate. The kinetics and localization of the enzyme were examined. The inhibition of the enzyme by the antimycotic agent, ‘15azasterol, was verified. -Introduction The biosynthetic pathways of ergosterol, the major sterol in yeast, and cholesterol include a A8i1* -sterol intermediate as a result of the demethylat~on of lanosterol at C-14 [l-3]. The A 14( ’ 5)-bend is reduced forming 4,4dimethylcholesta-8,14dienol, prior to the removal of the C-4 methyl groups and the modification of the side chain [4]. The presence of a A14-reductase, the enzyme involved in the saturation of the A14(15)-bond, was demonstrated in rat liver homogenate during cholesterol synthesis [ 51 and later observed in whole yeast cells during the conversion of 5~~rgosta-8,~4dien-3~-01 to ergosterol [ 11. Recently, an unusual Ag*14-sterol (ergosta-8,14-dien-3,&ol, ignosterol) was isolated from Saccharomyces cereuisiae after treatment with an antimycotic agent, 15-azasterol (15 -aza-24-methylene-D-homocholestadiene) E&71.

15-Azasterol * To whom correspondence

should be addressed.

Ignos terol

Removal of the 15-azasterol allowed the conversion of the ignosterol to ergosterol in vivo, confirming the necessity of a A’4-reductase. The accumulation of ignosterol provided an opportunity to assay directly for the Ai4-reductase activity in vitro. Materials and Methods Substrate preparation. Cultures of ~acc~aromyces cereuisiae 3701B grown ~aerobic~ly for 72 h at 28°C in 1% tryptone, 0.5% yeast extract, 2% glucose, were aerobically adapted in 0.1 M phosphate buffer (pH 5.8), 1% glucose, L-[Me-14C]methionine (0.5 PM, 5 /&i/l) and 200 fig/l 15-azasterol (15-aza-24methylene-D-homocholestadiene) and allowed to grow overnight at 28°C. The cells were harvested by centrifugation, acid-labilized, saponified, and the nonsaponifiable lipid fraction extracted as described previously [ 8,9]. This fraction was acetylated with pyridine and an excess of acetic anhydride and the acetylated sterols separated by thin-layer chromatography on 10% AgN03impregnated silica gel plates in cyclohexane/benzene (1 : 1, v/v). The major band was isolated and resaponified in methanolic KOH. The band was 98% ignosterol as determined by gas-liquid chromatography and ultraviolet analysis [7,9). The specific activity of the [‘4~]ignos~rol was calculated to be 4.4 . 10’ cpmlcrmol by methods previously described [lo]. Enzyme preparatt’on. Cultures of S. cerevisiae 3701B were grown anaerobically to prevent sterol synthesis in 1% tryptone, 0.5% yeast extract, 2% glucose for 48--72 h at 28°C. Cells were harvested in the cold under argon to maintain anaerobic conditions. The cells were then washed 3 times in cold distilled water and resuspended in 0.1 M citrate, 0.1 M phosphate buffer, adjusted to pH 6.0 (1 g wet weight cells/ml buffer). The resuspended cells were placed in a Duran flask with 0.25 mm glass beads (25 ml cell suspension/40 g beads) and broken by a 30-s burst in a Bronwill MSK cell homogenizer with COZ cooling. The glass beads and unbroken cells were removed by centrifugation at 2500 X g for 20 min. The whole cell extract was then run on a cold Sephadex column (G-75 coarse beads hydra~d in the above buffer) with a bed volume of 1 g dry weight/ml cell extract. The de-salted whole cell extract was collected and used as the standard enzyme preparation. Cellular fractionation. Cells were grown aerobically overnight at 28’C in 1% tryptone/0.5% yeast extract/2% ethanol. After harvesting, the cells were treated with 0.5 M ~-mercaptoeth~ol in 0.1 M Tris-HCl buffer (pH 9.3) at 2 ml/g wet weight cells for 5 min. After 2 washes with 0.44 M sucrose/l0 mM phosphate/l mM EDTA buffer (PI-I 5.8), the cells were resuspended in Helix pomatia intestinal juice (Glusulase)-buffer (1 : 4, v/v; Glusulase: above sucrose buffer) and incubated at 28°C with gentle shaking for 1 h. The spheroplasts were pelleted and washed twice in the same sucrose buffer before passing through a French pressure cell at 1000 lb[inch*. Cell debris was removed by repeated centrifugations at 2000 X g. The cell extract was then centrifuged at 17 000 Xg for 10 min. The supernatant fraction was used to prepare the non-mitochondrial fraction as described below (Scheme I). The mitochondrial fraction (pellet I) was resuspended in sucrose buffer, loaded onto 20-‘70% sucrose gradients and centrifuged at 50 000 Xg for 3 h in a Beckman SW-25.1

rotor on a Beckman Model L-Z ultracentrifuge. The resulting mitochondrial band was used for the mitochondrial fraction in the enzyme assay. The supe~at~t fraction from the above procedure was centrifuged 3 times at 35 000 Xg for 15 min to remove any remaining mitochondrial fragments. A portion of the 35 000 Xg supernatant fraction was centrifuged at 101 000 Xg for 1 h in a Beckman Type 40 rotor on a Beckman L-2 ultracentrifuge. The resulting fractions (‘floating lipid layer’, supernatant, soft pellet and hard pellet) were all assayed for enzyme activity (Scheme I). SPHEROPLASTS French press 2000 X g centrifugation 17 000 X g centrifugation

r-------------1

SUPERNATANT

PELLET I

50 000 I

MITOCHONDRIAL

1

ASSAY

X g centrifugation

I

35 000 X g centrifugation I SUPERNATANT II

BAND

1

ASSAY

101 000 X g centrifugation *

“Lipid layer” Supernatant III Soft pellet Hard pellet

1

ASSAY Scheme I. Schematic diagram depicting cellular fractionation procedure. Cells were grown aerobicaliy with ethanol as the primary energy source, and treated with glusulase to form spheroplasts. Differential centrifugations were employed to obtain the various cellular fractions. Standard enzyme assays were performed with aliquots from each fraction.

Enzyme assay. To assay the enzyme activity of the Ai4-reductase, [‘*Clignosterol was used as a substrate and the “C-labeled product formed in the enzyme reaction was recovered. The enzyme assays were performed in 2.7 ml 0.1 M citrate, 0.1 M phosphate buffer (pH 6.0), 0.1 ml 10 mM NADPH and 1 ml enzyme fraction (final concentration of protein 5 me/ml). Protein concentrations were determined by the method of Lowry et al. [ll]. [ “C]Ignosterol was added in 0.2 ml 100% ethanol. The reaction mixtures were incubated at 28°C under argon with gentle shaking for 2 h. The reaction rate during this period was linear. The reactions were terminated by addition of 10 ml 5% trichloroacetic acid and placed on ice for 10 min. The mixture was centrifuged at 500 X g for 15 min and the sterols were removed from the protein pellet by treatment with d~ethylsulfoxide [9] and extraction with n-hexane. The sterols were acetylated and separated by thin-layer chromato~phy as detailed above. The enzyme product was located about 14 cm above the baseline and ignosterol about 7 cm from the baseline. The bands were scraped and eluted

304

with methylene chloride and anhydrous diethyl ether through Na,SO, columns. The radioactivity of the samples was counted on a liquid scintillation spectrometer. Enzyme product identification. The sterol isolated as the product of the enzyme activity was analyzed by ultraviolet spectroscopy on a Cary Model 11 recording spectrometer and by coupled gas-liquid chromatography-mass spectroscopy as previously described [ 91. Materi&. Azasterol, designated A258223, was the generous gift of Eli Lilly and Co. Silica gel (HF-254) plates were produced by E.M. Laboratories and the solvents were from Mallinckrodt. L-[Me-‘4C]Methionine was from New England Nuclear. Na,SO, was purchased from J.T. Baker Co. and the cofactors were purchased from Sigma Chemical Co. Sephadex (G-75) was a product of Pharmacia; Glusulase was from Endo Laboratories, Inc. AgN03 was purchased from American Scientific and Chemical. Results The ignosterol derivative produced by the A14-reductase activity did not show a distinctive ultraviolet absorption maximum in hexane. Ignosterol shows spectroscopy of the a A,,, at 250 nm [7]. Gas-liquid chromato~apl~y-rn~s product {as an acetate) had a molecule ion peak at m/e 442 (100%) with other significant peaks at 311 (60%), 255 (48%) and 427 (15%). These correspond to a major ion with a mass equal to that of a singly unsaturated C-28 steryl acetate and to fragments with the loss of acetate plus 1 proton (-CZH402) plus part of the saturated side chain (-CSH,,), the loss of the acetate plus 1 proton (-C2H402) plus the complete side chain (-C9H19), and the loss of one methyl (-CH,), respectively. The enzyme product no longer contained the A8Y14-heteroannular conjugated diene as was shown by the ultraviolet absorption spectroscopy and mass spectroscopy. Gas chromatography-mass spectroscopy data of the derivative are consistent with an ergosta-B(or 7)-en-3@-ol acetate. Unfortunately, not enough of the derivative could be accumulated to attempt nuclear magnetic resonance. No other radioactively labeled products were found. The A“‘-reductase activity was optimal at a pH of 6.0 (Fig. 1) as was demonstrated by assays in buffers ranging from pH 5.0-8.0. Various ions were added, most of them having no effect. The exception was Mg*+, which decreased activity. Co~espondingly, the use of Sephadex columns to de-salt the enzyme preparation increased activity 5-fold. The addition of chelating agents, such as EDTA, slightly increased activity, but not to the extent of the Sephadex procedure. Of the cofactors tried (including NAD, NADH, NADP and ATP), NADPH gave the best stimulation, increasing enzyme activity 50-fold. NADP possibly due to its conversion to also increased activity approx , lo-fold, NADPH by enzymes in the whole cell extract. Enzyme solubilization by detergents like Triton X-100 decreased enzyme activity. No difference was seen between assays done under anaerobic conditions and assays done with oxygenation. The enzyme activity appears to be confined to the non-mitochondrial fraction (Table I). The greatest activity was in the hard protein pellet, suggesting

305

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1 53

, 60

I 65

I 70

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Fig. 1. Percent conversion of ignosterol by A 14-reduetase activity as a function of pH and buffer. Standard enzyme assays were performed with 1.5 I.IM 114Cl&nosterol. The buffer used in the assays and enzyme preparations were as follows: pH 5.0-6.5, 0.1 M citrate, 0.1 M phosphate adjusted to PH with KOH; pH 7.0-8.0.0.1 M Trls adjusted to pH with HCI. The greatest conversion was observed at pH 6.0. Fig. 2. Lineweaver-Burk plots of data from standard A 14-reductase assays. A*4-Reductase activity was non-competitively inhibited by the addition of 15.azasterol. V. [14C]ignosterol derivative formed in counts per minute (X1O-3): S, concentration of [ 14Clignosterol in &moJfl (X10T2). 4, control, without azasterol: a, azasterol, 22 nM: 0, azasterol, 13 &I.

that the enzyme is an insoluble protein. No further purification was done. For routine assays, lengthy purification steps to increase enzyme activity proved infeasible due to the lability of the enzyme. The apparent Km of the A14-reductase for ignosterol was determined to be 60 &I from Lineweaver-Burn plots (Fig. 2). The Km for 4,4dimethylcholes~E&14,24-trienol, which probably is the natural substrate of the A14-reductase in yeast, may be different because of interactions of the enzyme with the C-4 methyl groups. TABLE I DISTRIBUTION OF A’*-REDUCTASE

ACTSVITY IN VARIOUS CELLULAR FRACTIONS IN YEAST

CelJs were grown aerobically with ethanol as the primary energy source. 1 ml of each fraction was used as the enzyme preparation. Cellular fraction

Ignosterol converted (48 per mg protetn)

Mitochonddal band Supernatant II “Lipid layer” Supernatant III Soft pellet Hard pellet

1.4 21.6 3.5 4.6 24.2 43.1

306

The A14-reductase has been shown to be inhibited by 15azasterol in vivo at very low concentrations [ 61. Our in vitro data confirmed that 15-azasterol is an effective inhibitor of this enzyme, The Lineweaver-3urk plots indicated that the inhibition was non-competitive and that the fii was 2 nM (Fig. 2). Possible feed-back inhibition by ergosterol was also examined. Ergosterol at concentrations examined, up to 200 ,uM, did not show any inhibition of the enzyme activity (data unpublished). Discussion The reduction of the A*,14 -intermediate in the ergosterol biosynthetic pathway to a A’-intermediate has been shown to occur by the trans addition of two hydrogens to the 14a- and 15@-positions [12]. Our studies indicate that NADPH donates at least one of the hydrogens in this reaction, analogous to the reaction in rat liver cholesterol biosynthesis [ 131. Several reactions in ergosterol biosynthesis involved in modifying the side chain structure of the sterol were demonstrated to occur in the yeast mitochondrial fraction [ 10,141. Like some other ring structure transformations [ 15, 161, however, A14-reduction appears to take place in the cytoplasmic fraction. Ergosterol was not formed in this assay. It should be possible though, with slight alterations in the assay, to generate ergosterol from ignosterol in vitro as has already been done in vivo [6]. 15-azasterol, a potent antimycotic agent, has previously been shown to inhibit the Az4-transmethylase [17], the A24’28)-methylene reductase, and the A14-reductase [6,7]. Our results suggest that l5-azasterol inhibition of the Ai4-reductase activity is non-competitive with ignosterol. This is in contrast to the competitive inhibition demonstrated with the other two enzymes. The Ai4reductase displays a greater sensitivity to 15-azasterol than either of the other enzymes both in vivo and in vitro. 15-azasterol must have a high degree of specificity for the A14-reductase as very low concentrations (25 nM) block the reaction with ignosterot completely. The A14-reductase assay thus may be from useful in the future in identifying Ai -reductase mutants, p~icul~ly azasterol-resistant isolates. Acknowledgements This work was supported in part by grants from the National Science Foundation (PCM 76-11715) and the U.S. Public Health Service (AM-05190). C.B. was the recipient of an N.L. Tartar research fellowship. Oregon Agricultural Experiment Station technical paper No. 4865. References 1

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