Wabolism of A24-sterols by yeast mutants blocked in removal of the C-14 methyl group
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Degrnrrmenf ~fChemistry,Simon Fruser University, Buanahy, B . C . , Canada V5A PSQ Received January 30,1978
Pierce, A. M., Mueller, R. B., Unrau, A. M. & Oehlschlager, A. C. (1978) Metabolism of A2"sterols by yeast mutants blocked in removat of the C-14 methyl group. Carl. J . Biochem. 56, 794-880 We have investigated the metabolism of exogenously provided A"-sterols by whole celi cultures of a polyew-resistant mutant (D10) of Candida ai'bicuras blocked at removal of the C-14 methyl group. Comparison of the relative efficiencies of transmethylation at C-24 of selected rol sterol substrates revealed the following substrate preferences of the Cundida ~ i ~ ~ - s t emethyltransferase (EC 2.1.1.41): zymosterol > 4a-rnethylzymosteroB > I&-methy8zyrnosterol. Exogenous 4,4-dimethylzymosterol was not transrnethyiated by mutant D10. Incorporation of into the sterols of a I910 the 14C-labelledmethyl group of S-adenosyl-L-[methyl-14Cjmethionine culture preloaded with zyrnsstero8 indicated that zymcssterol was a better (40 x )i substrate than endogenous lanosterol. Feeding aymosterol to D18 and a polyene-resistant strain of Saccharc~rnpcescevevtsias (Nys-P1W) that was aiso blocked at removai s f the C-14 methyl group gave 24-methyl sterols possessing AZ2and ring B unsaturation. Mutant Dl0 was able to produce ergosterol from zymosterol whereas Nys-PI00 produced ergosta-7,22-dienol. When grown in the a known inhibitor of the A24-steroI methylpresence of 3 pM 25-am-24,25;-dihydrozymosterol, transferase, Nys-PI00 accumulated I&-methylzymosterol, a minor metabolite in this mutant under normal growth conditions and hitherto unidentified as a yeast sterol. Pierce, A. M., MueBler, R. B., Urahriu, A. M. & Oehlschlager, A. C. (1978) Metabolism of A24-sterolsby yeast mutants blocked in removal s f the C-14 methyl group. Csan. J . Bicrchsm. 56, 794-800 Nous avons etudie Be metabolisme de A24-sterolsexogenes par des cultures cellulaires entieres d'un mutant (DIO) de Caia~tidaalbicuns resistant aux polyenes et dans lequel I'enlevement du groupe mkthyle en (2-14 est bloque. La cornparaison des efficacites relatives de la transrnethylation sur le C-24 des sterols choisis rkvele les prefkrences suivantes d e la dZ4-sterol mkthyltransferase (EC 2.1.1.41) de Candidu: zymosterol > 4-mCthyizymosterol > I&-methylzyn~osterol.Le 4,4-dimethylzymosterol exogkne n9est pas transrnethyle par le mutant D10. L'incorporation du groupe rnithyle marque au 14C de la S-adenosyl-L-[methyB-14G]methionine dans les sterols d'une culture de Dl8 prealhlement psurvue de zymosterol mnntre que le zymsstCrol est un meilleur substrat (40 X ) que le lanosterol endogene. La presence d e zyrnosterol dans fescultures du D10eB d'une ssuche de Saccharomyces cerevisiae (Nys-P106)),resistante aux polyenes et dans Iaquelle l'enlevement du groupe mkthyle en C-14 est aussi bloque, donne les 24-mkthylsterols non saturees en C-22 et dans 19anneauB. Le mutant D l 0 est capable de fabriquer 19ergosteroHa partir du zymosterol tandis que le Nys-PI00 fabrique l'ergosta-7,22-diknol. CultivCe en pksence de 3 ph4 de 25-aza-24,25;-dihydrozyrraoster~~1, un inhibiteur connu de la AD-sterol rnethyltransfkrase, ie Nys-PI80 accurnule Be 14a-mCthylzyrnostero1, un metabolite minew dans ce mutant croissant dans des conditions normales et, jusqu'ici, non identifik comme un sterol de la levure. [Traduit par le journal]
Inbrodlmebfsn previous investigations of the biosynthetic conversion oflanosterol (1, see Fig. 11 to ergosterol (239 in yeast have been aided by the isolation of polyene-resistant strains that accumulate ergosterol precursors (I-5). Complete sterol analyses of wild-type and mutant strains (1-69 coupled with incorporation studies of labelled sterol pre-
cursors ( 8 , 4, '7, 8) have supported the view that the trdRsf0rmationsinvolved in the latter stages of ergosterol biosynthesis may occur in several alternative sequences (Fig. I). Genetic analysis of Saccharomyces cerevisia~ at steps in the a x ~ ~ e of~ desmethyl intermediates to ergosterol revealed that each enzymatic t~nsformationcould be attributed to one gene (9). Recently, we ( 5 ) and others (109 have described ABBREVIATIONS: 24-SMT, AZ4-sterol methyitransferase. ~ ' Y mutants ~ of~ C ~ n~d i d n~ that ~ ac-~ glpc, gasliquid partition chromatography; TMS, trimethylsilyi~ P RRT's, relative retention times; &Ims, gas chromatography - cumulate predominantly 24-methylene-24,25-dih~dro mass spectroscopv; id, internal diameter; m r . nuclear mag,,lanosterd (29 and smdl amounts of sbtusifoliol (4) and tie res6nance; tic; thin-layer ekna*omatograph;; NSF('S), con- lanosterol (1). The sterol composition of these mutants saponifiahle fraction(s); 25-AZ, 25-aza-24,25-dihydrozyrno- strongly suggests a block in the demethylase for removal sterol; 14a-rnethylzymosterol, B4a-methylcholesta-rB,24-dienol. of one k - and (or) the lb-methyl group(s). The
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PIERCE ET A L .
Fro. 1. General scheme for sterol biosynthesis in yeast.
predominance of 24-methylene-24,25- dihydrolanosterol (2) in these mutants indicates the ability of the 24-SMT (EC 2.1.1.41) of Candidn to accept lanosterol as a prirnary substrate when no 4.14-desmethyl sterols are present. Under nosmall circumstances, 2 represents a very minor component of Cundida sterols (4,5). Sprinson and co-workers ( I 1) have recently reported a mutant of S. c ~ r e v i s i ablocked ~ a t the removal of the 14a-methyl group which accumulates 14a-methylergosta-8,24628)-dienol (6). This is surprising since i~ \.itro studies of the 24-SMT system of wild-type S. cerevisiae (12) have indicated that lanosterol does not serve as a substrate and that 4,4-dimethyl- and &-methylzymosterol serve as poor substrates for this enzyme selative to zymosteroI. It would appear that under normal
circumstances the presence of a 14a-methyl group renders a ~ i ~ ~ - s t eunacceptable rol but that when no desmethyl sterols are available 14a-methyl sterols can act as substrates for the Saccharomyces 24-SMT. The absence of 14a-methyl sterols that possess Ax-, A9-, and A5*7-unsaturation in wild-type yeast suggests that I4a-methyl sterols are unacceptable substrates for the enzymes mediating these sterol transformations. Mutants lacking 14a-demethylase activity are of interest in the study of yeast sterol biosynthesis in that the transformations of 14a-desrnethyl sterols exogenously administered to these organisms may be monitored without complications due to endogenous sterol production. In the present study, we have investigated the metabolism of exogenously provided ~ % ~ ~ - s t eby r s whole ls cell
7%
CAN. J. BIOCHEM. VOL. 56, 1948
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cultures s f S. cerevisdae and C. ~lbichilnsm u t a n t s ckaracterized b y a block in l h - d e m e t h y l a s e activity. In t h e Catzdiclcr mutant, w e c o m p a r e d t h e relative efficiencies of transmetkyIation at @-24 o f selected sterol s u b s t r a t e s with t h a t of t h e endogenous substrate, lanosterol.
Isolation of44a-MethylzymosderoI A 18-mi inoculurn of Nys-PI00 statically incubated at 28°C for 24 h was added t o 200 ml of complete liquid medium (5) containing 3 pM 25-AZ, an inhibitor of the 24-SMT (13), and the culture was grown at 25°C with stirring for 48 h. The 200-ml culture was used t o inoculate 2&of complete medium which contained 3 p M 25-AZ, and the ctalture was grown in a 4-l Aask on a Virtis Materials and Methods fermenter at 28°C with 400 rpm stirring and 3 elmin aeration for I~tstrsararentatioazand Sterol Anaiy.~ds 48 h. The 25-AZ, synthesized in a previous investigation (13), Sterois were analyzed by gipc a s both the TMS and acetate was added as a 5 mhf solution in ethanol to the autoclaved derivatives using a gas chromatograph equipped with a flame medium. An equivalent amount of ethanol was added to control ionization detector. Peak areas were computed electronically. cultures. TMS derivatives were formed by addition of Tri-Sil/TBT The yeast were harvested by centrifugation, washed three (Pierce Chemical Co.) t o the free sterols. Acetates were formed times with distilled water, and saponified (1). The free sterols of by treatment with gyridine - acetic anhydride (1: 1, vlv) for 12h control and 25-AZ inhibited cultures were separated by tlc, and at room temperature. Analyses of TMS derivatives were per- analysis of the recovered bands by gBpc and ms gave the sterols formed on a 32.5 m x 0.25mm id glass column coated with listed in Table 1. Wecrystallization several times from methanoi OV-101 at 240°C. Acetates were analyzed both on the above of the 4-demethyl bands of control and 25-AZ inhibited cultures column a t 245°C and on a J.68rn x 2.2mm id glass column gave 1b-methylergosta-$,24(28)-dienol 66) and I&-methylpacked with 3% SSBLAR-1OC on Gas Chrom Q (16)0/120) at zymosteroi (5) respectively, both greater than 9% pure by glpc. 220°C. The identity of individual sterols was ascertained by ' at m/e 412 with the base peak The mass spectrum of 6 gave M comparison of glpc RRT's using 3P-cholestanol as an internal at m/e 397 (M+-CW,). The fragmentation pattern conformed standard with RRT9s previo~~sly determined for yeast sterols closely to that of the authentic sterol (I 1, 15). The RRT for the (13). The amounts of individual sterols were determined by acetate of 6 on OV-BOB capillary glpc agreed with published comparison of peak areas with that of the internal standard. results (16). Mass spectra were obtained on a mass spectrometer interfaced The major 4-demethyl sterol of the 25-AZ inhibited cultures t o a gc/ms data processor using an ionization voltage of 8$Oe&' was assigned the structure of 14a-methylzymosterol (5) on the and probe temperature of B8O"C. Coupled gclms was performed basis of the following physical data: mp 103.8-104.5"C; nmr on a gas chromatogiiph interfaced through a Watson-Biemann (CDCl,) 8: 0.72 (s, 3H, C-18 H), 0.89 (s, 3H, C-32 H), 0.90 (d, separator t o the ion source. Sterol acetates analyzed by gclms Jc-zoH, C-z EI 6.0 H Z , 3If, (2-21 HI, 0.95 (s, 3H, C-19 H), 1.60 and were separated on a 1-83m x 2.2 mm id glass column packed 1.68 (s and s , 3H and 3H, C-26 H and (2-27 W),3.57 (m,BH, C-3 with 3% SILAR-1OG on Gas Chrom Q (100/120) at 230°C. The H), 5.08 (m, BH. C-24 H); ms mle (rel. int., 9%): M+ 398 (71.9), amount of ergosterol was determined by ultraviolet spectros- 383 (loo), 365 (20.5), 313 (11.4), 271 (12.5), 245 (16-519231 (40.6), copy with hexane as the solvent. The nmr spectra were recorded 219 (25.0), 213 (13.3), 201 (15.5), 187 (18.7), 161 (23.81, 159 at 23°C using CDCI, a s the soevent and tetramethylsilane as the (18.9), 149 (13.4), 84'7 (15.8), 145 (16.8), 137 (12.9, 135 (13.4)internal standard. Melting points were determined on a calib- 133 (16.51, 131 (13.4), 123 (14.6), 121 (17.31, 119 (23.01, 109 rated apparatus. (40.8), 104 (26.8), 185 (23.8), 95 (38.91, 93 (22.8), 91 (18.2). 83 The tlc was carried out on plates (20 X 20cm) coated with (14.5), 81 (27.6). 79 (13.4), 69 (42.7), 67 (la.$), 55 (26.69, 43 silica gel HF-254 -+ 366 (Merck, type 68) of 0.5-mm thickness (11 .5),41 (22.8). containing 0.2% Rhodamine 6G. For initial separation of the NSF, tlc plates were developed once in methylcyclohexane - Steroi Feeding Experinaents Zymosterol, k-methy8zymostero1, 4,4-dimethylzymosterol, ethyl acetate (3:8, viv) to give 4,4-dimethyl, &-methyl, and 4-deanethyl sterol fractions. For the separation of sterol ace- obtusifo!iol, and lanosterol were available from previous investates, 25% AgNO, was added to the silica gel. Ail plates were tigations (7, 17). 1k-Methy8zymosterol was isolated from preconditioned at 120°C for 30 min before use. After visualiza- 25-AZ treated cultures s f Nys-PI00 (see above). The organisms were grown in lOOml of complete liquid tion under ultraviolet Bight, free sterols were eluted from the silica gel with hexane - diethyl ether (4: 1, vlv) and sterol ace- medium in 3W-nal Erlenmeyer flasks. The individual sterol tates were eluted from the AgN0,-silica gel with diethyl ether. (5 mg) or a mixture of two sterols (5 mg each) and 80mg of Solvents were routinely distilled or were of analytical reagent Tween 80 were dissolved in 0.3 ml of ethanol and added to the autoclaved medium. A 24-h 10-mi inocultam was added. and the grade. Radioactivity was assayed with a liquid scintillation culture was grown at room temperature (-25°C) under air atmsspectrometer. Samples were dissolved in a cocktail containing sphere with magnetic stirring well into stationary growth phase 4 g of PPO and 50 mg of POPOP per litre of toluene. A11 instru- (42-120h). The cultures were harvested by centrifugation, washed three times with distilled water, and saponified. The ments used were as previously described (5,7). NSF's were analyzed by glpc and ultraviolet spectroscopy and Yeast Strains and Maintenance the data reported in Tables 2 and 3. The polyene-resistant mutant Dl0 of C. albicapls was derived by chemical mutagenesis in a previous study (5). The S. cere- IncorporcrPion 43-Aden~syl[raze~l-'~C]~nethiunine rrisiae mutant designated Nys-3 was available from a previous A 2-t Erlenmeyer flask was prepared with 600 ml of complete study (1) and was found to be resistant to 3 8 U nystatin per medium and 30mg zymosterol, inoculated with a 24-h 6@ml rnillilitre. The strain Nys-PI00 was isolated at 180 U nystatin per culture of D 10, and grown at 25°C with stirring under air atmarnillilitre after successive reculturing of Nys-3 on Szybalski gra- sphere, After % h, the culture was harvested by centrifugation and washed twice with 0.1 M potassium phosphate buffer (pH dient plates (14) containing nystatin (5). The isolates were maintained on agar slopes s f complete 6.4). A 2.3-g portion ofcells was removed for saponification as a medium (5) at 7°C and were recultured at 3- to 4 w e e k intervals. eontrol. The remaining 15.9g of yeast were resuspended in In order to prevent revertant growth, nystatin was incorporated $418 ml of 0.1 ,%i potassium phosphate buffer containing 4% @uinto the slopes at 200 U per millilitre for D l 0 and 100 U per cose (wEv>and stirred magnetically at 25'C under air atmosphere for 5 min. S-adenosyl-L-[methyl- l4C]methionine (54 rnCilmmo1 millilitre for Nys-PI MI.
797
PIERCE ET AL. = 37GBq); International Chemical and Nuclear Corp.) in 1 mi s f ethanol was then added to give a final concentration of 8.125pCilrnl. and incubation with stirring proceeded for 0.5 h. After this time, cells were immediately harvested by centrifugation, washed twice in phosphate buffer, and saponified. After preliminary separation of the free sterols by tlc, the recovered sterol fractions were acetylated and further separated by AgNQ-tlc. The plates were developed once in benzene for the Cdemethyl fraction (I) or once in benzene-hexane (3.5:1, vlv) for both the 4,4-dimethyl and 4a-methyl fractions (4). Rhodarnine was removed frsm the recovered sterol acetates as previously described (7). To the recovered band containing ergost-7-en01 acetate (17-Ac) and ergosta-7.22-dienol acetate (19-Ac) were added 60mg each of unlabelled 17-Ac and 19-As. The 1%Ac and 19-Ac were then separated on AgWB, plates with one development in benzene-hexane 1:2 (vlv) followed by two developments in benzene-hexane 1:3.5 [vlv). To the recovered ergosterol acetate (21-Ae) and 24-rnstkylene-24.25-dihydrolinostero! acetate (2-As) were added respectively unlabelled 21-Ac (75 mg) and 2-Ac (50 mg); and the 2-Ac, 1%Ac, 19-Ac, and 21-Ae were recrystallized from methanol-chloroform to constant activity (Table 4). The 2 was isolated frsm mutant DIO grown in liquid culture as previously described (3, 17-Ac and 19-Ac were available from a previous study (7), and 21 was purchased from Eastman Kodak Co. Since other cold traps were not available, the remaining sterol acetates were assayed for radioactivity directiy.
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( 1 Ci
Results and Discussion A S. cerevisiae isolate designated Nys-PI00 (Table 1) was derived from the previously described (1) Nys-3 strain by successive reculturing on media containing increasing concentrations of raystatin. The isolate accumulated major quantities (Table 1) of f k-methylergosta8,24(28)-dienol(6). lanosterol (I),and squalene and small amounts sf obtusifoliol(4), 4,14-dimethylzymosterol(3), 24-methylene-24,25-dihydrolanosterol (2), and the hitherto unknown 14a-methylzymosterd (5). Its sterol composition was similar to that of the nystatin-resistant ,561 mutant recently described by Trocha et s l . (1 1 ) except that the Batter two sterols (2 m d 51 were not reported for SC1. Alkylatisn at C-24 of lanosterol has not been previously demonstrated in S . eerevisias. Identification of 1401-methylzymosterol(5) was carried out on samples isolated from Nys-P1BO treated with a known (13) 24-SMT inhibitor, 25-AZ. It could be deduced that ifthe bissynthetic sequence were f -,3+ 5 4 6 in this mutant, removal of 24-SMT from action should cause 5 to accumulate. At 3 pM 25-AZ, almost complete inhibition of 24-SMT occurred and 5 became the major sterol metabolite (Table 1). The identity of 5 was determined by examination s f its nmr and mass spectra. The C-lO@ and @-83P methyl groups of 5 absorbed at 6 0.95 and 0.72 respectively, in good agreement with the chemical shifts reported for 4,l4-dimethylzymosterol(11,17). The mass spectrum of 5 revealed the molecular ion peak at m/e 398 (6j28H460) with base peak mle 383 (M+-15). The base peak M+- 15 is characteristic s f sterols possessing a 14a-methyl group (15). Other intense peaks were m/e 365 [M+-(CW, H20)] and m/e 23 1 which arises by cleavage through ring D and loss of a 14a-methyl group (15). Since Nys-PlOO was derived from Nys-3, a mutant
+
TABLE 1. Sterol production in S. cereoisiae mutant Nys-PI00 grown in the presence of 25-AZ
25-AZ, pM
3
0 Dry cell weight, g/C Sterols, % of dry cell weighta Squalene, % of dry cell weightu Sterd composition, T Lanasterol (1)
24-Methylene-24,25-dihydrolanosterol(2) 4,14-Bimethylzymosterol (3) Obtusifolioi (4) 14a-Methylzymosterol (5) 14cw-Methylergosta-8,24(28)-dienoB (6)
4.34 0.57 0.21 37.7 0.5 4.5 13.1 1.4 42.7
3.16
0.52 -h
39.0 ND" 11.5 C
49.8 0.3
ODetermined by gIpc, bNot determined. CPercentageof the total sterol pool. W N , not detected. e < 0.1%.
TABLE 2. Metabolism of aymostersl by whole cell culture of Nys-9100"
A
Sterol compssition Lanosterol (1)
24-Methylene-24,25-dihydr~Ianosterol (2) 4,14-Dimethylzymostero~ (3) Obtusifbliol(4) l4a-Niethylzymosterol (5)
14m-Methyle~gssta-8~24(28)-dieno1 (6)
O ~ B
34.0 1.6 2.4 12.5 1.1 19.0
Zyrnosterol (11) Ckolesta-7,24-dienol (12) Fecostersl (13) Ergosta-8,22-diensl (14) Epiaterol (16) Ergost-7-enol (1%)
Ergosta-7,22,24(28)-trienol (18) Ergssta-7,224ienol (19) Ergosterol (21)
0.7
4.3 0.4 2.0 0.3 0,2 0.3 21,2 C
aNys-P1OOwas grown in the presence o f zymosterol. bPercenPageof the total sterol pool. = < 0.1%.
blocked at the As'6)-dehydrsgenase that accumulates ergosta-7,22-dienol, it was of interest to determine if Nys-PI00 alss contained this biock. Accordingly, we cultured Nys-PI00 in the presence of ayrnosterol. Sterol intermediates characteristic of Nys-3 (1) appeared in this experiment (Table 2) indicating that all sterol modifying enzymes except the C-14 demethylase and AS6)-deRydrogenase are functional in Wys-P100. Mutant SG I (11) was alss isolated from a strain of S. cereviaiae that was partially blocked at the A5(6'-dehydrogenase. In addition to the S. eerevisiae isolate that lacked the C-14 demethylase, we have generated a mutant of C . ulbieuns that has decreased C-4 demethylase activity and lacks the C-14 demethylase. This mutant (D10) surprisingly accumulates 24-methylene-24,25-dihydrolanosterol (2) as its major (85%) sterol metabolite. As with Nys-PfOO, exogenous zymosterol was incorporated into Dl0 cultures but was converted to ergosterol (Table 3). A small amount of cholesta-7,24-dienol (12) was de-
CAN. I. BICPCHEM. VOL. 56. 1978
TABLE 3. MetabsIism of AS4-sterolsby whole cdl cultures of C. calba'cansmutant Dl8
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Sterol composition,
Sterol fed
Grits
None Lanosterol 1 4,84-Dimethylzymssterol 3 4,4-Dirnethylzy1nostero17 4a-Methylzyrnsstersl 9 14a-MethyIzymostef.sl5 Zymosterol 18 4a-Methylzyrnssterol and 143-methylzyn~c~sker01 9&5
4 2 2
7
8
9
PO
NDb NB NB 6.0
3 2
7.0 74.1 6.5 72.8 33.8 41.1 ND 5.5 76.2 0.5 10.8 N D 51.2 13.5 17.6 2 . 9 ND
ND ND ND ND ND ND NB
ND ND ND 2.0 6.0 ND ND
ND ND ND 0.2 0.4 ND NB
3
32.4 41.7
ND 3.9 0.5
1
2
3
4
5
6
11
21
ratio
--
--
-
-
3
3
4.0 85.3 8.7 79.6
0.4 10.3 8.6 11.1 5.1 13.8 0.6 10.7 5.7 6 . 9
5.9
8.0 N D
-
UPercentape of the total sterol pool. Values are means for the number of experiments indicated. *KD,n ~ detected. t cP
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Degrnrrmenf ~fChemistry,Simon Fruser University, Buanahy, B . C . , Canada V5A PSQ Received January 30,1978
Pierce, A. M., Mueller, R. B., Unrau, A. M. & Oehlschlager, A. C. (1978) Metabolism of A2"sterols by yeast mutants blocked in removat of the C-14 methyl group. Carl. J . Biochem. 56, 794-880 We have investigated the metabolism of exogenously provided A"-sterols by whole celi cultures of a polyew-resistant mutant (D10) of Candida ai'bicuras blocked at removal of the C-14 methyl group. Comparison of the relative efficiencies of transmethylation at C-24 of selected rol sterol substrates revealed the following substrate preferences of the Cundida ~ i ~ ~ - s t emethyltransferase (EC 2.1.1.41): zymosterol > 4a-rnethylzymosteroB > I&-methy8zyrnosterol. Exogenous 4,4-dimethylzymosterol was not transrnethyiated by mutant D10. Incorporation of into the sterols of a I910 the 14C-labelledmethyl group of S-adenosyl-L-[methyl-14Cjmethionine culture preloaded with zyrnsstero8 indicated that zymcssterol was a better (40 x )i substrate than endogenous lanosterol. Feeding aymosterol to D18 and a polyene-resistant strain of Saccharc~rnpcescevevtsias (Nys-P1W) that was aiso blocked at removai s f the C-14 methyl group gave 24-methyl sterols possessing AZ2and ring B unsaturation. Mutant Dl0 was able to produce ergosterol from zymosterol whereas Nys-PI00 produced ergosta-7,22-dienol. When grown in the a known inhibitor of the A24-steroI methylpresence of 3 pM 25-am-24,25;-dihydrozymosterol, transferase, Nys-PI00 accumulated I&-methylzymosterol, a minor metabolite in this mutant under normal growth conditions and hitherto unidentified as a yeast sterol. Pierce, A. M., MueBler, R. B., Urahriu, A. M. & Oehlschlager, A. C. (1978) Metabolism of A24-sterolsby yeast mutants blocked in removal s f the C-14 methyl group. Csan. J . Bicrchsm. 56, 794-800 Nous avons etudie Be metabolisme de A24-sterolsexogenes par des cultures cellulaires entieres d'un mutant (DIO) de Caia~tidaalbicuns resistant aux polyenes et dans lequel I'enlevement du groupe mkthyle en (2-14 est bloque. La cornparaison des efficacites relatives de la transrnethylation sur le C-24 des sterols choisis rkvele les prefkrences suivantes d e la dZ4-sterol mkthyltransferase (EC 2.1.1.41) de Candidu: zymosterol > 4-mCthyizymosterol > I&-methylzyn~osterol.Le 4,4-dimethylzymosterol exogkne n9est pas transrnethyle par le mutant D10. L'incorporation du groupe rnithyle marque au 14C de la S-adenosyl-L-[methyB-14G]methionine dans les sterols d'une culture de Dl8 prealhlement psurvue de zymosterol mnntre que le zymsstCrol est un meilleur substrat (40 X ) que le lanosterol endogene. La presence d e zyrnosterol dans fescultures du D10eB d'une ssuche de Saccharomyces cerevisiae (Nys-P106)),resistante aux polyenes et dans Iaquelle l'enlevement du groupe mkthyle en C-14 est aussi bloque, donne les 24-mkthylsterols non saturees en C-22 et dans 19anneauB. Le mutant D l 0 est capable de fabriquer 19ergosteroHa partir du zymosterol tandis que le Nys-PI00 fabrique l'ergosta-7,22-diknol. CultivCe en pksence de 3 ph4 de 25-aza-24,25;-dihydrozyrraoster~~1, un inhibiteur connu de la AD-sterol rnethyltransfkrase, ie Nys-PI80 accurnule Be 14a-mCthylzyrnostero1, un metabolite minew dans ce mutant croissant dans des conditions normales et, jusqu'ici, non identifik comme un sterol de la levure. [Traduit par le journal]
Inbrodlmebfsn previous investigations of the biosynthetic conversion oflanosterol (1, see Fig. 11 to ergosterol (239 in yeast have been aided by the isolation of polyene-resistant strains that accumulate ergosterol precursors (I-5). Complete sterol analyses of wild-type and mutant strains (1-69 coupled with incorporation studies of labelled sterol pre-
cursors ( 8 , 4, '7, 8) have supported the view that the trdRsf0rmationsinvolved in the latter stages of ergosterol biosynthesis may occur in several alternative sequences (Fig. I). Genetic analysis of Saccharomyces cerevisia~ at steps in the a x ~ ~ e of~ desmethyl intermediates to ergosterol revealed that each enzymatic t~nsformationcould be attributed to one gene (9). Recently, we ( 5 ) and others (109 have described ABBREVIATIONS: 24-SMT, AZ4-sterol methyitransferase. ~ ' Y mutants ~ of~ C ~ n~d i d n~ that ~ ac-~ glpc, gasliquid partition chromatography; TMS, trimethylsilyi~ P RRT's, relative retention times; &Ims, gas chromatography - cumulate predominantly 24-methylene-24,25-dih~dro mass spectroscopv; id, internal diameter; m r . nuclear mag,,lanosterd (29 and smdl amounts of sbtusifoliol (4) and tie res6nance; tic; thin-layer ekna*omatograph;; NSF('S), con- lanosterol (1). The sterol composition of these mutants saponifiahle fraction(s); 25-AZ, 25-aza-24,25-dihydrozyrno- strongly suggests a block in the demethylase for removal sterol; 14a-rnethylzymosterol, B4a-methylcholesta-rB,24-dienol. of one k - and (or) the lb-methyl group(s). The
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PIERCE ET A L .
Fro. 1. General scheme for sterol biosynthesis in yeast.
predominance of 24-methylene-24,25- dihydrolanosterol (2) in these mutants indicates the ability of the 24-SMT (EC 2.1.1.41) of Candidn to accept lanosterol as a prirnary substrate when no 4.14-desmethyl sterols are present. Under nosmall circumstances, 2 represents a very minor component of Cundida sterols (4,5). Sprinson and co-workers ( I 1) have recently reported a mutant of S. c ~ r e v i s i ablocked ~ a t the removal of the 14a-methyl group which accumulates 14a-methylergosta-8,24628)-dienol (6). This is surprising since i~ \.itro studies of the 24-SMT system of wild-type S. cerevisiae (12) have indicated that lanosterol does not serve as a substrate and that 4,4-dimethyl- and &-methylzymosterol serve as poor substrates for this enzyme selative to zymosteroI. It would appear that under normal
circumstances the presence of a 14a-methyl group renders a ~ i ~ ~ - s t eunacceptable rol but that when no desmethyl sterols are available 14a-methyl sterols can act as substrates for the Saccharomyces 24-SMT. The absence of 14a-methyl sterols that possess Ax-, A9-, and A5*7-unsaturation in wild-type yeast suggests that I4a-methyl sterols are unacceptable substrates for the enzymes mediating these sterol transformations. Mutants lacking 14a-demethylase activity are of interest in the study of yeast sterol biosynthesis in that the transformations of 14a-desrnethyl sterols exogenously administered to these organisms may be monitored without complications due to endogenous sterol production. In the present study, we have investigated the metabolism of exogenously provided ~ % ~ ~ - s t eby r s whole ls cell
7%
CAN. J. BIOCHEM. VOL. 56, 1948
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cultures s f S. cerevisdae and C. ~lbichilnsm u t a n t s ckaracterized b y a block in l h - d e m e t h y l a s e activity. In t h e Catzdiclcr mutant, w e c o m p a r e d t h e relative efficiencies of transmetkyIation at @-24 o f selected sterol s u b s t r a t e s with t h a t of t h e endogenous substrate, lanosterol.
Isolation of44a-MethylzymosderoI A 18-mi inoculurn of Nys-PI00 statically incubated at 28°C for 24 h was added t o 200 ml of complete liquid medium (5) containing 3 pM 25-AZ, an inhibitor of the 24-SMT (13), and the culture was grown at 25°C with stirring for 48 h. The 200-ml culture was used t o inoculate 2&of complete medium which contained 3 p M 25-AZ, and the ctalture was grown in a 4-l Aask on a Virtis Materials and Methods fermenter at 28°C with 400 rpm stirring and 3 elmin aeration for I~tstrsararentatioazand Sterol Anaiy.~ds 48 h. The 25-AZ, synthesized in a previous investigation (13), Sterois were analyzed by gipc a s both the TMS and acetate was added as a 5 mhf solution in ethanol to the autoclaved derivatives using a gas chromatograph equipped with a flame medium. An equivalent amount of ethanol was added to control ionization detector. Peak areas were computed electronically. cultures. TMS derivatives were formed by addition of Tri-Sil/TBT The yeast were harvested by centrifugation, washed three (Pierce Chemical Co.) t o the free sterols. Acetates were formed times with distilled water, and saponified (1). The free sterols of by treatment with gyridine - acetic anhydride (1: 1, vlv) for 12h control and 25-AZ inhibited cultures were separated by tlc, and at room temperature. Analyses of TMS derivatives were per- analysis of the recovered bands by gBpc and ms gave the sterols formed on a 32.5 m x 0.25mm id glass column coated with listed in Table 1. Wecrystallization several times from methanoi OV-101 at 240°C. Acetates were analyzed both on the above of the 4-demethyl bands of control and 25-AZ inhibited cultures column a t 245°C and on a J.68rn x 2.2mm id glass column gave 1b-methylergosta-$,24(28)-dienol 66) and I&-methylpacked with 3% SSBLAR-1OC on Gas Chrom Q (16)0/120) at zymosteroi (5) respectively, both greater than 9% pure by glpc. 220°C. The identity of individual sterols was ascertained by ' at m/e 412 with the base peak The mass spectrum of 6 gave M comparison of glpc RRT's using 3P-cholestanol as an internal at m/e 397 (M+-CW,). The fragmentation pattern conformed standard with RRT9s previo~~sly determined for yeast sterols closely to that of the authentic sterol (I 1, 15). The RRT for the (13). The amounts of individual sterols were determined by acetate of 6 on OV-BOB capillary glpc agreed with published comparison of peak areas with that of the internal standard. results (16). Mass spectra were obtained on a mass spectrometer interfaced The major 4-demethyl sterol of the 25-AZ inhibited cultures t o a gc/ms data processor using an ionization voltage of 8$Oe&' was assigned the structure of 14a-methylzymosterol (5) on the and probe temperature of B8O"C. Coupled gclms was performed basis of the following physical data: mp 103.8-104.5"C; nmr on a gas chromatogiiph interfaced through a Watson-Biemann (CDCl,) 8: 0.72 (s, 3H, C-18 H), 0.89 (s, 3H, C-32 H), 0.90 (d, separator t o the ion source. Sterol acetates analyzed by gclms Jc-zoH, C-z EI 6.0 H Z , 3If, (2-21 HI, 0.95 (s, 3H, C-19 H), 1.60 and were separated on a 1-83m x 2.2 mm id glass column packed 1.68 (s and s , 3H and 3H, C-26 H and (2-27 W),3.57 (m,BH, C-3 with 3% SILAR-1OG on Gas Chrom Q (100/120) at 230°C. The H), 5.08 (m, BH. C-24 H); ms mle (rel. int., 9%): M+ 398 (71.9), amount of ergosterol was determined by ultraviolet spectros- 383 (loo), 365 (20.5), 313 (11.4), 271 (12.5), 245 (16-519231 (40.6), copy with hexane as the solvent. The nmr spectra were recorded 219 (25.0), 213 (13.3), 201 (15.5), 187 (18.7), 161 (23.81, 159 at 23°C using CDCI, a s the soevent and tetramethylsilane as the (18.9), 149 (13.4), 84'7 (15.8), 145 (16.8), 137 (12.9, 135 (13.4)internal standard. Melting points were determined on a calib- 133 (16.51, 131 (13.4), 123 (14.6), 121 (17.31, 119 (23.01, 109 rated apparatus. (40.8), 104 (26.8), 185 (23.8), 95 (38.91, 93 (22.8), 91 (18.2). 83 The tlc was carried out on plates (20 X 20cm) coated with (14.5), 81 (27.6). 79 (13.4), 69 (42.7), 67 (la.$), 55 (26.69, 43 silica gel HF-254 -+ 366 (Merck, type 68) of 0.5-mm thickness (11 .5),41 (22.8). containing 0.2% Rhodamine 6G. For initial separation of the NSF, tlc plates were developed once in methylcyclohexane - Steroi Feeding Experinaents Zymosterol, k-methy8zymostero1, 4,4-dimethylzymosterol, ethyl acetate (3:8, viv) to give 4,4-dimethyl, &-methyl, and 4-deanethyl sterol fractions. For the separation of sterol ace- obtusifo!iol, and lanosterol were available from previous investates, 25% AgNO, was added to the silica gel. Ail plates were tigations (7, 17). 1k-Methy8zymosterol was isolated from preconditioned at 120°C for 30 min before use. After visualiza- 25-AZ treated cultures s f Nys-PI00 (see above). The organisms were grown in lOOml of complete liquid tion under ultraviolet Bight, free sterols were eluted from the silica gel with hexane - diethyl ether (4: 1, vlv) and sterol ace- medium in 3W-nal Erlenmeyer flasks. The individual sterol tates were eluted from the AgN0,-silica gel with diethyl ether. (5 mg) or a mixture of two sterols (5 mg each) and 80mg of Solvents were routinely distilled or were of analytical reagent Tween 80 were dissolved in 0.3 ml of ethanol and added to the autoclaved medium. A 24-h 10-mi inocultam was added. and the grade. Radioactivity was assayed with a liquid scintillation culture was grown at room temperature (-25°C) under air atmsspectrometer. Samples were dissolved in a cocktail containing sphere with magnetic stirring well into stationary growth phase 4 g of PPO and 50 mg of POPOP per litre of toluene. A11 instru- (42-120h). The cultures were harvested by centrifugation, washed three times with distilled water, and saponified. The ments used were as previously described (5,7). NSF's were analyzed by glpc and ultraviolet spectroscopy and Yeast Strains and Maintenance the data reported in Tables 2 and 3. The polyene-resistant mutant Dl0 of C. albicapls was derived by chemical mutagenesis in a previous study (5). The S. cere- IncorporcrPion 43-Aden~syl[raze~l-'~C]~nethiunine rrisiae mutant designated Nys-3 was available from a previous A 2-t Erlenmeyer flask was prepared with 600 ml of complete study (1) and was found to be resistant to 3 8 U nystatin per medium and 30mg zymosterol, inoculated with a 24-h 6@ml rnillilitre. The strain Nys-PI00 was isolated at 180 U nystatin per culture of D 10, and grown at 25°C with stirring under air atmarnillilitre after successive reculturing of Nys-3 on Szybalski gra- sphere, After % h, the culture was harvested by centrifugation and washed twice with 0.1 M potassium phosphate buffer (pH dient plates (14) containing nystatin (5). The isolates were maintained on agar slopes s f complete 6.4). A 2.3-g portion ofcells was removed for saponification as a medium (5) at 7°C and were recultured at 3- to 4 w e e k intervals. eontrol. The remaining 15.9g of yeast were resuspended in In order to prevent revertant growth, nystatin was incorporated $418 ml of 0.1 ,%i potassium phosphate buffer containing 4% @uinto the slopes at 200 U per millilitre for D l 0 and 100 U per cose (wEv>and stirred magnetically at 25'C under air atmosphere for 5 min. S-adenosyl-L-[methyl- l4C]methionine (54 rnCilmmo1 millilitre for Nys-PI MI.
797
PIERCE ET AL. = 37GBq); International Chemical and Nuclear Corp.) in 1 mi s f ethanol was then added to give a final concentration of 8.125pCilrnl. and incubation with stirring proceeded for 0.5 h. After this time, cells were immediately harvested by centrifugation, washed twice in phosphate buffer, and saponified. After preliminary separation of the free sterols by tlc, the recovered sterol fractions were acetylated and further separated by AgNQ-tlc. The plates were developed once in benzene for the Cdemethyl fraction (I) or once in benzene-hexane (3.5:1, vlv) for both the 4,4-dimethyl and 4a-methyl fractions (4). Rhodarnine was removed frsm the recovered sterol acetates as previously described (7). To the recovered band containing ergost-7-en01 acetate (17-Ac) and ergosta-7.22-dienol acetate (19-Ac) were added 60mg each of unlabelled 17-Ac and 19-As. The 1%Ac and 19-Ac were then separated on AgWB, plates with one development in benzene-hexane 1:2 (vlv) followed by two developments in benzene-hexane 1:3.5 [vlv). To the recovered ergosterol acetate (21-Ae) and 24-rnstkylene-24.25-dihydrolinostero! acetate (2-As) were added respectively unlabelled 21-Ac (75 mg) and 2-Ac (50 mg); and the 2-Ac, 1%Ac, 19-Ac, and 21-Ae were recrystallized from methanol-chloroform to constant activity (Table 4). The 2 was isolated frsm mutant DIO grown in liquid culture as previously described (3, 17-Ac and 19-Ac were available from a previous study (7), and 21 was purchased from Eastman Kodak Co. Since other cold traps were not available, the remaining sterol acetates were assayed for radioactivity directiy.
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( 1 Ci
Results and Discussion A S. cerevisiae isolate designated Nys-PI00 (Table 1) was derived from the previously described (1) Nys-3 strain by successive reculturing on media containing increasing concentrations of raystatin. The isolate accumulated major quantities (Table 1) of f k-methylergosta8,24(28)-dienol(6). lanosterol (I),and squalene and small amounts sf obtusifoliol(4), 4,14-dimethylzymosterol(3), 24-methylene-24,25-dihydrolanosterol (2), and the hitherto unknown 14a-methylzymosterd (5). Its sterol composition was similar to that of the nystatin-resistant ,561 mutant recently described by Trocha et s l . (1 1 ) except that the Batter two sterols (2 m d 51 were not reported for SC1. Alkylatisn at C-24 of lanosterol has not been previously demonstrated in S . eerevisias. Identification of 1401-methylzymosterol(5) was carried out on samples isolated from Nys-P1BO treated with a known (13) 24-SMT inhibitor, 25-AZ. It could be deduced that ifthe bissynthetic sequence were f -,3+ 5 4 6 in this mutant, removal of 24-SMT from action should cause 5 to accumulate. At 3 pM 25-AZ, almost complete inhibition of 24-SMT occurred and 5 became the major sterol metabolite (Table 1). The identity of 5 was determined by examination s f its nmr and mass spectra. The C-lO@ and @-83P methyl groups of 5 absorbed at 6 0.95 and 0.72 respectively, in good agreement with the chemical shifts reported for 4,l4-dimethylzymosterol(11,17). The mass spectrum of 5 revealed the molecular ion peak at m/e 398 (6j28H460) with base peak mle 383 (M+-15). The base peak M+- 15 is characteristic s f sterols possessing a 14a-methyl group (15). Other intense peaks were m/e 365 [M+-(CW, H20)] and m/e 23 1 which arises by cleavage through ring D and loss of a 14a-methyl group (15). Since Nys-PlOO was derived from Nys-3, a mutant
+
TABLE 1. Sterol production in S. cereoisiae mutant Nys-PI00 grown in the presence of 25-AZ
25-AZ, pM
3
0 Dry cell weight, g/C Sterols, % of dry cell weighta Squalene, % of dry cell weightu Sterd composition, T Lanasterol (1)
24-Methylene-24,25-dihydrolanosterol(2) 4,14-Bimethylzymosterol (3) Obtusifolioi (4) 14a-Methylzymosterol (5) 14cw-Methylergosta-8,24(28)-dienoB (6)
4.34 0.57 0.21 37.7 0.5 4.5 13.1 1.4 42.7
3.16
0.52 -h
39.0 ND" 11.5 C
49.8 0.3
ODetermined by gIpc, bNot determined. CPercentageof the total sterol pool. W N , not detected. e < 0.1%.
TABLE 2. Metabolism of aymostersl by whole cell culture of Nys-9100"
A
Sterol compssition Lanosterol (1)
24-Methylene-24,25-dihydr~Ianosterol (2) 4,14-Dimethylzymostero~ (3) Obtusifbliol(4) l4a-Niethylzymosterol (5)
14m-Methyle~gssta-8~24(28)-dieno1 (6)
O ~ B
34.0 1.6 2.4 12.5 1.1 19.0
Zyrnosterol (11) Ckolesta-7,24-dienol (12) Fecostersl (13) Ergosta-8,22-diensl (14) Epiaterol (16) Ergost-7-enol (1%)
Ergosta-7,22,24(28)-trienol (18) Ergssta-7,224ienol (19) Ergosterol (21)
0.7
4.3 0.4 2.0 0.3 0,2 0.3 21,2 C
aNys-P1OOwas grown in the presence o f zymosterol. bPercenPageof the total sterol pool. = < 0.1%.
blocked at the As'6)-dehydrsgenase that accumulates ergosta-7,22-dienol, it was of interest to determine if Nys-PI00 alss contained this biock. Accordingly, we cultured Nys-PI00 in the presence of ayrnosterol. Sterol intermediates characteristic of Nys-3 (1) appeared in this experiment (Table 2) indicating that all sterol modifying enzymes except the C-14 demethylase and AS6)-deRydrogenase are functional in Wys-P100. Mutant SG I (11) was alss isolated from a strain of S. cereviaiae that was partially blocked at the A5(6'-dehydrogenase. In addition to the S. eerevisiae isolate that lacked the C-14 demethylase, we have generated a mutant of C . ulbieuns that has decreased C-4 demethylase activity and lacks the C-14 demethylase. This mutant (D10) surprisingly accumulates 24-methylene-24,25-dihydrolanosterol (2) as its major (85%) sterol metabolite. As with Nys-PfOO, exogenous zymosterol was incorporated into Dl0 cultures but was converted to ergosterol (Table 3). A small amount of cholesta-7,24-dienol (12) was de-
CAN. I. BICPCHEM. VOL. 56. 1978
TABLE 3. MetabsIism of AS4-sterolsby whole cdl cultures of C. calba'cansmutant Dl8
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Sterol composition,
Sterol fed
Grits
None Lanosterol 1 4,84-Dimethylzymssterol 3 4,4-Dirnethylzy1nostero17 4a-Methylzyrnsstersl 9 14a-MethyIzymostef.sl5 Zymosterol 18 4a-Methylzyrnssterol and 143-methylzyn~c~sker01 9&5
4 2 2
7
8
9
PO
NDb NB NB 6.0
3 2
7.0 74.1 6.5 72.8 33.8 41.1 ND 5.5 76.2 0.5 10.8 N D 51.2 13.5 17.6 2 . 9 ND
ND ND ND ND ND ND NB
ND ND ND 2.0 6.0 ND ND
ND ND ND 0.2 0.4 ND NB
3
32.4 41.7
ND 3.9 0.5
1
2
3
4
5
6
11
21
ratio
--
--
-
-
3
3
4.0 85.3 8.7 79.6
0.4 10.3 8.6 11.1 5.1 13.8 0.6 10.7 5.7 6 . 9
5.9
8.0 N D
-
UPercentape of the total sterol pool. Values are means for the number of experiments indicated. *KD,n ~ detected. t cP
