5747 $800 (pk), [CXIPP~-2657"; A%" 228 nm, 281, and 299 sh (log E 4.55, 4.36, and 4.06); mass spectrummk 606 (M+h 400, 293, 236, 220, 2% (base), 192, and 91; high-resolution mass spectrum m/e 605.2992 (calcd for M+ - 1 = C ~ ~ H ~ L N ~m/e OZ, 605.3014).
Acknowledgments. This research was supported by
Grant CAI 1450 from the National Institutes of Health to M. S. The authors are grateful to Professor M. Tomita for a sample of authentic 2,10-dimethoxyaporphine. B. baluchistanica was identified by Dr. A. R. Beg, Plant Taxonomist, Forest Research Institute, Peshawar, Pakistan.
Biosynthesis of Ergosterol in Yeast. Multiple Pathways'
Evidence for
M. Fryberg, A. C. Oehlschlager,* and A. M. Unrau Contribution from the Department of Chemistry, Simon Fraser University, Burnaby 2, British Columbia, Canada. Received February 17, 1973
Abstract: The conversion of lanosterol to ergosterol in Saccharomyces cerevisiae has been investigated. Timecourse analysis of the sterol content and feeding-trapping experiments with suspected intermediates led to the discovery of several alternative pathways in the latter stages of ergosterol biosynthesis. Maintenance of the yeast under anaerobic conditions depleted the sterol content of the organism. The sterols most rapidly consumed under these conditions were those possessing A5v7unsaturation. During anaerobic maintenance squalene accumulated. A subsequent change to aerobic conditions was accompanied by accelerated sterol production. Timecourse analysis of the changing sterol composition during aeration indicated that the initial structural modifications following the formation of lanosterol involved nuclear demethylation at C4 and C14 as well as alkylation at C24. In order to investigate subsequent modifications synthesis of suspected 4,14-desmethyl-24-alkyl sterol intermediates (unlabeled and l4C or 3H labeled) possessing varied unsaturation, e.g., As, A7, A5v7, and A 2 4 ( 2 8 ) , was carried out. Chromatographic separation and analysis of S. cerevisiae sterol mixtures led to the discovery of five previously unreported sterols (14 and 16-19) in this organism. Feeding and trapping experiments with suspected intermediates and previously reported yeast sterols revealed that several alternative pathways are operative in the latter stages of the lanosterol to ergosterol bioconversion. Based on relative incorporation efficiencies, the major route from fecosterol to ergosterol involves A8 +. A7 isomerization, introduction of unsaturation at CW,then at C5,and finally reduction of the 24-methylene. The first of these transformations was shown to be reversible while the last three were essentially nonreversible.
T
he enzymatic conversion of lanosterol (1) to ergosterol (2) in yeast (Saccharomyces cerevisiae) requires six general transformations: (1) removal of the three methyl groups in lanosterol at C, and CI4; (2) alkylation at C2, with concomitant reduction at C26and generation of a A24(28)-methylene; (3) isomerization of the A8 double bond to A'; (4) reduction of the A24(28)double bond generating a Cza-methyl; (5) introduction of a A Z 2double bond; and ( 6 ) introduction of a A5 double bond.
HO
m
6
7
HO
A priori, these transformations could occur in any order. A variety of studies, particularly those involving investigation of enzyme-substrate specifications, have provided an insight into the approximate order in which these transformations occur. By analogy with (1) Preliminary communication: M. Fryberg, A. C. Oehlschlager, and A. M. Unrau, Biochem. Biophys. Res. Commun., 48, 593 (1972). (2) L. J. Mulheirn and P. J. Ramm, Chem. SOC.Reu., 27,259 (1972).
8
cholesterol biosynthesis, it has been assumed that nuclear demethylation is the initial step in the lanosterol to ergosterol conversion. Gaylor, et al., have recently provided evidence that demethylation precedes AB+ A7 Fryberg, et al. / Biosynthesis of Ergosterol in Yeast
5748
d
HO
HO
J
!
8
i
HO
I HO
HO
HO
&
KT
h.
HO
HO
&’
a@=+ HO
Figure 1. Model for proposed routes to ergosterol in yeast.
isomerization. They4 have also demonstrated that zymosterol (3) is superior to 4,4-dimethylzymosterol (4), 4a-rnethylzymosterol (5), cholesta-7,24-dien-3& 01 (6), cholesta-5,24-dien-3/3-ol (7), and cholesta-5,7,24-trien-3/3-01 (8) as a substrate for a soluble A Z 4 sterol methyltransferase isolated from yeast (Figure (3) (a) J. T. Moore, Jr., and J. L. Gaylor, Arch. Biochem. Biophys.,
124, 167 (1968); (b) J. L. Gaylor, C. V. Delwiche, and A. C . Swindell, Steroids, 8, 353 (1966). (4) J. T. Moore, Jr., and J. L. Gaylor, J . B i d . Chem., 245, 4684 (1970).
1). Assuming the relative rates observed in this system reflect in vivo substrate specificities, C2 I alkylation most likely occurs after partial as well as complete nuclear demethylation, but prior to introduction of the AZ2,A7,and A3 unsaturation. Consistent with this sequence is the apparent absence in yeast sterol mixtures of 6-8 and the presence of 4,b 5 , 5 9,F zymosterol,’ ( 5 ) G. Ponsinet and G. Ourisson, Bull. SOC.Chim. Fr., 3682 (1965). (6) D. H. R. Barton, D. M. Harrison, G. P. Moss, and D. A. Widdowson, J . Chem. SOC.,775, (1970). (7) I. Smedley-MacLean, Biochem. J., 22, 22 (1928).
Journal of the American Chemical Society / 95:17 / August 22, 1973
5749 Scheme I
AcO
AcO
AcO
24
RO
14 R=H 14-AC RsAc
AcO
22
AcO
23
& 15-AC
fecosterol (lo),* episterol (11),* 5,6-dihydroergosterol (12),9 and ergosta-5,7,22,24(28)-tetraen-3/3-01 (13).1° Since zymosterol is the best substrate for the AZ4-sterol methyltransferase and the immediate product of its enzymatic methylation is its Cza-niethylene derivative (fecosterol), a rather well-defined pattern is evident to this point. Subsequent to this, As + A' isomerization, A: 4 ( 2 8 ) reduction, and Ab and A ? ? unsaturation occur in an undefined order. Figure 1 shows the possible alternative sequences that could conceivably operate in the transformation of fecosterol to ergosterol. Using Figure 1 as a model, we approached the investigation of these alternative pathways from three complementary directions. Initially we synthesized the suspected sterol intermediates 10-20. With the synthetic samples in hand, we proceeded with the isolation of most of the corresponding natural sterols from yeast sterol mixtures. In order to obtain information on possible in viuo precursor-product relationships, we investigated the time course change in sterol composition of anaerobically pretreated, aerobically growing yeast. Although these experiments were designed to minimize initial sterol content and maximize sterol production in the cultures under study, little information concerning the interconversions of the 4,14desmethyl sterols was obtained. Significantly more light was shed on the operative in vivo interconversions by incubation of the synthetic intermediates with aerobically growing yeast. This was coupled with assays for radioactivity in suspected metabolites (nearest structural neighbors) as well as ergosterol. It was found that all but two of the suspected sterols, namely ergosta-8,22,24(28)-trien-3/3-01 (15) and ergosta-5,7-
dien-3/3-01 (20), were present in growing yeast and involved in the production of ergosterol. Chemical Synthesis of Intermediates. The preparation of 3p-acetoxyergosta-8,22-diene(14-Ac) and 3pacetoxyergost-8-ene (16-Ac) was carried out according to previously reported procedures. Thus, oxidation of 3/3-acetoxyergosta-7,22-diene (17-Ac) with mercuric acetate gave the 7,9(11),22-triene 21" which was epoxidizedl? at the 9(11) unsaturation (22) and con(23) with verted to 3~-acetoxyergosta-8,22-dien-ll-one ferric chloride13 (Scheme I). Reduction of 23 with lithium aluminum hydride gave the dioli4 which was converted to 14 with lithium in ethylamine. Acetylation of 14 gave 14-Ac. Ozonolysis of 14-Ac followed by a Wittig reaction'& of the resultant aldehyde (24) gave 36-acetoxyergosta8,22,24(28)-triene (15-Ac). Experiments': executed prior to this work have revealed that the ozonolysisWittig sequence used for conversion of 14-Ac to 15-Ac did not alter the configuration at C-20. Although mixtures of A ? ? cis and trans isomers are undoubtedly formed in the Wittig reactions, the proportion of cis isomer can be kept to a minimum by use of nonpolar solvents such as hexane.16 Nuclear magnetic resonance spectra and thin layer chromatographic analysis of the crystallized Wittig products revealed that substantially pure trans isomers could be isolated by one or two crystallizations of the crude reaction product. l F (1 1) G. Saucy, P. Geistlich, R. Helbling, and H. Heusser, Helc. Chim. Acta, 37, 250 (1954).
(12) H. Heusser, I80 % ergosterol and tetraenol (13) with only minor amounts of lanosterol, zymosterol, and other intermediates. The first source proved to be suitable for the isolation of a greater variety of suspected intermediates. Partially purified sterol mixtures obtained from mother liquors (major portion of ergosterol previously removed) were further chromatographically separated into five major fractions as illustrated in Figure 2. Fraction 1 contained squalene, identified by comparison of its nmr spectrum with an authentic sample, and a clear oil consisting of a number of low-boiling compounds which had glpc retention times close to that of squalene. Fraction 2 contained essentially pure lanosterol (1). Fraction 3 contained a mixture of 4,4-dimethylzymosterol (4), 4a-methylzymosterol (5), and 4-methyl24(28)-methylenezymosterol (9) whose structures were assigned by comparison of glpc retention times and mass spectralg with published values. Fraction 4 was a mixture of mono-, di-, and triunsaturated 4,14-desmethyl sterols. Fraction 5 contained ergosterol (2), tetraenol (13), and a third sterol. Fraction 4 was acetylated and further separated by chromatography. Three subfractions were collected and further separated by preparative thin layer chromatography as illustrated in Figure 3. The acetates of 3, 10, 11, 12, 14, 16, 17, and 18 were identified in this fraction. Separation of fraction 5 gave ergosterol (2), tetraenol (13), and A5,7,24(28)trienol(l9). Thus, in addition to those sterols previously rep~rted,~-lO14 and 16-19 also occur in s. cerevisiae. Barton, et al.,'O have recently reported the isolation of 17-19 from the same source. The only sterols in Figure 1 not yet detected in this yeast are 15 and 20. (18) G . W, Patterson, Anal. Chem., 43, 1165 (1971). (19) T. J. Scallen, A. K. Dhar, and E. D. Loughran, J . B i d . Chem., 246, 3168 (1971). (20) D. H. R. Barton, U. M. Kempe, and D. A. Widdowson, J . Chem. SOC.,Perkin Trans. I , 513 (1972).
Journal of the American Chemical Society 1 95:17 1 August 22, 1973
5751
d
0
,,A,
-
AcO
I
FRACTION 1. SOUALENE t OIL FRACTION 2. LANOSTEROL
DIETHYL
Figure 2.
31
'
R=AC
R=BZ
FRACTION 4 (ACETATES1
%
COLUMN CHROMATOGRAPHY ON ALUMINA
32
RO
A g N g /SILICA GEL COLUMN
+
FRACTION 3. 4.4-DIMETHVL4 a- METHYL STEROLS FRACTION 4. MONO-, DI- AND TRIUNSATURATED DESMETHVL STEROLS FRACTION 5. TRI- AND TETRAUNSATURATED STEROLS
BENZENE
-100
--
% DIETHYLETHER
t
t UV(-I
0 U V ( t 1 230,240mp
ERGOST-7-EN ACETATE ERGOST-8-EN ACETATE
BENZENE
Column chromatography of yeast sterols.
Results and Discussion Time-Course Study of Yeast Sterol Composition. As a prelude to the investigation of precursor-product relationships of the intermediates present in yeast sterol mixtures, we studied conditions under which vigorous sterol synthesis could be induced. Ideally a yeast culture was desired which was depleted of sterols and which upon initiation of sterol production would generate sequentially each intermediate. It was hoped that from such time course studies of the sterol composition would emerge a biogenetic sequence. Several experiments indicated inoculation of liquid medium with yeast followed by aerobic growth gave cultures in which the amount of sterol on a dry weight basis remained relatively unchanged over several days. Time course analysis of the sterol composition of these cultures revealed rather insignificant variations that were not interpretable in terms of precursor-product relationships. Yeast harvested during this type of growth contained ergosterol (2) and tetraenol (13) as major (80z) sterols with minor amounts of zymosterol (3) and lanosterol (1) also present. It was known from the experiments by Klein, et aZ.,21 that sterol formation could be induced in Saccharomyces cerevisiae by vigorous aeration of cells that had previously been maintained under strictly anaerobic conditions. Under these conditions, S. cerevisiae becomes auxotropic for sterols and squalene accumulates.22 This is considered to be due to the necessity of molecular oxygen for the formation of (21) H. P. Klein, N. R. Eaton, and J. C. Murphy, Biochim. Biophys. Acta, 13, 591 (1954). (22) H. P. Klein, J. Bacterial,, 73, 530 (1957).
ERGOSTA-7.22-DIEN ERGOSTA-8.22-OIEN
1
ACETATE ACETATE
ZVMOSTEROL ACETATE
ERGOSTA -7.22,24(281TRIEN ACETATE
J I J l FECOSTEROL ACETATE EPISTEROL ACETATE HEXANEIBENZENE 6:4 (DEVELOPED 2x1
Figure 3.
Fractionation of desmethyl sterol acetates.
2,3-oxidosqualene, the immediate precursor of lanosterol. 3 , 2 4 We considered it likely that aeration of such a culture would be accompanied by an accumulation of primordial intermediates. Depending on the rates of interconversion, some intermediates could appear more prominantly in the sterol mixture during early growth rather than at later stages. When the yeast was cultured under nitrogen for 80 hr, the major portion (95 %) of the sterols disappeared. The composition of the small amount of sterol left had changed appreciably. Although there were traces of ergosterol, the major sterol was ergosta-7,22-dien3/3-01 (12) followed by decreasing amounts of ergosta7,22,24(28)-trien-3p-o1 (17), lanosterol (l),zymosterol (3), and ergosta-8,22-dien-3@-01(14). The anaerobically pretreated yeast was resuspended in fresh medium and aerated. Upon aeration the total sterol content increased rapidly (Figure 4). The (23) E. J. Corey, W. E. Russey, and P. R. Ortiz De Montellano, J . Amer. Chem. SOC.,88, 4750 (1966). (24) E. E. van Tamelen, J. D. Willett, R. B. Clayton, and K. E. Lord, ibid., 88, 4752 (1966).
Fryberg, et at. 1 Biosynthesis of Ergosterol in Yeast
5752
.
...............A?. ...............
130&
I
&;
*.......~.~ ........ ,,s.__._..__....... no ~
~
t l
1
I
A
/
I
/?
8
12
10
18
24
TIME (HOURS)
Figure 4. Variation of sterol composition in aerobically growing yeast.
changing composition of the yeast sterol mixture during this period is illustrated in Figure 4. These time-course studies revealed that squalene concentration increased briefly upon the commencement of aeration and then decreased sharply as the total sterol content increased. When sterol production became constant, squalene synthesis approximately paralleled sterol synthesis. Three types of behavior with respect to sterol pool size were revealed. The concentrations of lanosterol, 4-methylated and 4,424(28)-trienol19 dimethylated intermediates, and increase rapidly to attain early maxima, then subsequently decrease to low values. This confirms previous r e p 0 r t s ~ ~ concerned ,~6 with the accumulation of lanosterol during fermentation. The concentrations of the As and A7 4,14-desmethyl sterols increase slowly toward their steady-state concentrations. Episterol ( l l ) , ergosta-7,22,24(28)-trien3p-01 (17), and fecosterol (10) increase steadily with their relative concentrations deviating only slightly from a 4 : 2 : 1 ratio. Noteworthy is the relatively low concentration of ergost-7-en-3p-01 (18) compared with the other desmethyl sterols. The concentrations of ergosterol and tetraenol (13) increase slowly at first, then at increasing rates through the middle of the time course. Finally, these two sterols reach high concentrations in the sterol fraction as would be expected of final metabolic products. The variation of concentration with time observed for the 4,lCdimethylated and 4-methyleted intermediates is that expected of initial intermediates produced from squalene in a culture primed by squalene accumulation. Since these compounds are undoubtA5s77
(25) W. I
