Eur. J. Biochem.

246, 518-529 (1997)

0FEBS 1997

Identification of cDNAs encoding sterol methyl-transferases

involved in the second methylation step of plant sterol biosynthesis
Pierrette BOUVIER-NAVE’,Tania HUSSELSTEIN’, Thierry DESPREZ2and Pierre BENVENISTE’
’ Institut de Biologie MolCculaire des Plantes, DCpartement d’Enzymologie Cellulaire et MolCculaire, Institut de Botanique, Strasbourg, France
Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Versailles, France

(Received 3 February 1997) - EJB 97 0158/2

Two methyl transfers are involved in the course of plant sterol biosynthesis and responsible for
the formation of 24-alkyl sterols (mainly 24-ethyl sterols) which play major roles in plant growth and
development. The first methyl transfer applies to cycloartenol, the second one to 24-methylene lophenol.
Five cDNA clones encoding two Arabidopsis thalianu, two Nicotiana tabacum and one Ricinus communis
S-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SMT) were isolated. The deduced amino
acid sequences of A. thaliana and N. tabacum SMT are about 80% identical in all possible combinations.
In contrast they are about 4 0 % identical with the deduced amino acid sequence of R. communis SMT
and the published Glycine max sequence. Both A. thaliana and one N. tabacum SMT cDNAs were
expressed in a yeast null mutant erg6, deficient in AdoMet zymosterol C24-methyltransferase and contain-
ing C24-non-alkylated sterols. In all cases, several 24-ethylidene sterols were synthesized. A thorough
study of the sterolic composition of erg6 expressing the A. thaliana cDNA 411 (erg6-4118-pYeDP60)
showed 24-methylene and 24-ethylidene derivatives of 4-desmethy1, 4a-methyl and 4,4-dimethyl sterols
as well as 24-methyl and 24-ethyl derivatives of 4-desmethyl sterols. The structure of 5a-stigmasta-8, Z-
24(24’)-dien-3P-o1, the major sterol of transformed yeasts, was demonstrated by 400 MHz ’H NMR.
Microsomes from erg-6-4218-pYeDP60 were shown to possess AdoMet-dependent sterol-C-methyl-
transferase activity. Delipidated preparations of these microsomes converted cycloartenol into 24-methy-
lene cycloartanol and 24-methylene lophenol into 24-ethylidene lophenol, thus allowing the first identifi-
cation of a plant sterol-C-methyltransferase cDNA. The catalytic efficiency of the expressed SMT was
17-times higher with 24-methylene lophenol than with cycloartenol. This result provides evidence that
the A. thuliana cDNA 41 1 (and most probably the 3 plant SMT cDNAs presenting 80% identity with it)
encodes a 24-methylene lophenol-C-24l methyltransferase catalyzing the second methylation step of plant
sterol biosynthesis.
Keywords: plant sterol methyltransferase; yeast transformation; complementation analysis; 24-methylene
lophenol ; cycloartenol.

Sterols from fungi and higher plants differ from vertebrate
sterols by the presence of an extra alkyl group at C24 [l, 21.
Whereas most fungi sterols possess a methyl group at C24,
higher plants contain both 24-methyl and 24-ethyl sterols. This
alkylation of the side chain is catalyzed by S-adenosyl-L-methio-
nine (AdoMet) sterol C-methyltransferases (SMT). In Saccharo-
myces cerevihiue, the SMT converts zymosterol (IX) into feco-
sterol (XVI) [3] (Fig. 3). In higher plants, the presence of 24-
ethyl sterols results from two distinct methyl transfers from Ad-
oMet [l, 21. According to the chemical structures of intermedi-
Correspondence to P. Bouvier-NavC, Institut de Biologie Moltculaire
des Plantes, DCpartement d’Enzymolagie Cellulaire et MolCculaire,
Instituf de Botanique, 28 rue Goethe, F-67083, Strasbourg CCdex, France
Fax: +33 03 88 35 84 84.
URL: http :llibmp.u-strasb~.~rl
Abbreviations. SMT, sterol C-methyltransferase; AdoMet, S-adeno-
sylmethionine.
Enzymes. S-Adenosyl-L-methionine: zymosterol C24-methyltrans-
ferase (EC 2.1.1.41) is the yeast SMT [3] encoded by ERG6 (32-341.
Note. The nucleotide sequences reported here have been submitted
to the GenBanEMBL data bank and are available under accession
numbers: cDNA 411, X89867; cDNA 205, U71400; cDNA 132,
U71108; cDNA 412, U71107; cDNA rmt, U81313.
ates of plant sterol biosynthesis and substrate-specificity studies,
it is generally assumed that cycloartenol (I) (Fig. 1) is the sub-
strate of the first methylation reaction, resulting in 24-methylene
cycloartanol (11) [4-71, whereas 24-methylene lophenol (IV) is
the preferred substrate for the second methylation, yielding 24-
ethylidene lophenol (V) [8, 91 (Fig. 1). Because the chemical
structures of I and IV are very different, it has been suggested
that the two methylation reactions would be catalyzed by two
different enzymes [9]. However, since no plant SMT has been
purified so far, the hypothesis of a unique plant SMT catalyzing
both alkylations [lo] should be considered. In any case the sec-
ond methylation is a unique process, absent in vertebrates and
most fungi, leading to the higher plant 24-ethyl sterols. These
typical phytosterols were shown to develop specific interactions
with plant phospholipids [ l l ] .
Two plant SMT genes were recently cloned and their gene
products preliminarily characterized [ 12, 131. The first reported
plant SMT cDNA was isolated from Glycine rnax [12, 141; the
deduced amino acid sequence showed three conserved regions
found in AdoMet-dependent methyltransferases and 47 % iden-
tity with the predicted amino acid sequence of ERC6, the yeast
SMT-encoding gene. The G. max cDNA was expressed in
Escherichia coli and shown to possess SMT activity: in the pres-
 Bouvier-NavC et al. ( E m J. Biochem. 246) 519

I n I11

IV V

1111
11
24-methyl sterols 24-ethyl sterols
Fig. 1. Simplified sterol biosynthesis pathway in higher plants showing the two methylation steps. AdoHCy, S-adenosylhomocysteine;
I, cycloartenol = 4,4,14a-trimethyl-9~,19-cyclo-5a-cholest-24-en-3~-o1;
11, 24-methylene cycloartanol = 4,4,14a-trimethyl-9P,19-cyclo-5u-ergost-
24(24')-en-3&01; 111, obtusifoliol = 4a,l4a-dimethyl-5a-ergosta-8,24(24')-dien-3~-01; IV, 24-methylene lophenol = 4a-methyl-5a-ergosta-
7,24(24')-dien-3P-ol;V, 24-ethylidene lophenol = 4a-methy1-5a-stigmasta-7,Z-24(24')-dien-3~-01.

ence of AdoMet, the cell-free extract of the transformed E. coli
converted lanosterol (VI) to 24-methylene lanosterol (XI)
(Fig. 3) [12]. The second described plant SMT cDNA was iso-
lated from Arabidopsis thaliana in our laboratory [13]; its se-
quence also contained features typical of methyltransferases but
showed only 38% identity with ERG6 This cDNA, termed
cDNA 411, was used to transform a wild type S. cerevisiae as
well as the yeast null mutant erg6, which is deficient in the yeast
SMT zymosterol C24-methyltransferase ; in both cases, several
24-ethyl and 24-ethylidene sterols were synthetized, indicating
that the cDNA 411 encodes a plant SMT able to perform two
sequential methylations at C24 and C24' of the yeast sterols
[13]. At this stage we could not identify exactly the enzyme
encoded by ORF 4118. It could be (a) a cycloartenol-C24-meth-
yltransferase (SMT, in Fig. 1) of low substrate specificity, (b) a
24-methylene-lophenol-C241-methyltransferase (SMT, in Fig. 1)
of low substrate specificity, or (c) a single SMT able to perform
both methylation reactions.
We now report the characterization of the enzymatic product
of ORF 4118 expressed in the yeast null mutant erg6. A sub-
strate-specificity study clearly showed that 24-methylene lophe-
no1 (IV) is the preferred substrate, thus ruling out hypotheses (a)
and (c). Furthermore, the cloning of other SMT cDNAs from
A. thaliana, Nicotiana tabacum and Ricinus communis and the
comparison of their deduced amino acid sequences with those
of SMT cDNAs from Glycine max 1121 and A. thaliana [I31
showed that they are distributed into two distinct groups, one
including the R. communis and G. max cDNAs and the other,
the A. thaliana and N. tabacum cDNAs. Since all the results
presented here clearly show that the second group most probably
encodes a 24-methylene lophenol C24'-methyltransferase, the
first group is suggested to encode a cycloartenol C24-methyl-
transferase.
EXPERIMENTAL PROCEDURES
Strains, media and culture conditions. Escherichia coli.
XLlblue recA- [recAJ, lac-, endAI, gyrA96, thi, hsdRI7,
SupE44, relAl, (F'proAB, laclq, lacZAMJ5, TnlO)].Saccharo-
myces cerevisiae. erg6 (a) ade5 his7-2 leu2-3,112 ura3-52 ERG6
d::LEU2; erg2 (a) ade5 his7-2 leu2-3,112 ura3-52 ERG2-
4 : :LEU2.
Strains transformed with pYeDP6O were grown for 3 days at
30°C on minimum medium YNB [6.7 g/l yeast nitrogen base,
(Difco)] containing suitable supplements (50 pg/ml each). The
culture was centrifuged, the pellet resuspended in a complete
medium [ l o g/l yeast extract (Difco), 10 g/l bactopeptone
(Difco), 20 g/l galactose] and grown overnight at 30°C.
Plasmids. The plasmid pYeDP6O [15] was used to transform
yeast strains. This plasmid contains an E. coli replication origin,
a yeast 2 pm plasmid replication origin, an E. coli ampicillin-
resistance gene, and the yeast genes URA3 and ADE2. It utilizes
an expression cassette including a galactose-inducible hybrid
promoter and a phosphoglycerate kinase (PGK) terminator.
Gene expression is driven by the upstream activating sequence
of the yeast GAL10 and CYC4 genes.
cDNA libraries. The clone VBVEC07 EMBL: emb]
Z342031ATTS 3237 was isolated by the systematic screening of
a cDNA library (Versailles - VB) from in vitro-grown, 5-day-
old, etiolate seedlings of Arabidopsis thaliana ecotype Columbia
[16]. As shown in results, VBVEC07 was used as template in
PCR experiments to obtain cDNA 205.
cDNA 41 1 was isolated from an A. thaliana ecotype Colum-
bia siliques library constructed in Lambda Zap I1 (Stratagene)
by Giraudat et al. [17]. The cloning site was EcoRI.
cDNAs 132 and 412 were isolated from a library of 3-week-
old N. tabacum variety Xanthi line SH6 calli derived from leaf
520 Bouvier-Navt et al. (Eul: J. Biochern. 246)
protoplasts. The library was prepared by one of us with Lambda
Zap I1 (Stratagene). cDNAs were cloned unidirectionally in
EcoR1, XIzoI.
cDNA rmt was isolated from a library of endosperm and
embryo of immature castor fruits (R. communis strain Baker
296). This library was made in Lambda Zap I1 by van de Loo
et al. [18] and cDNAs were cloned in EcoRI, XhoI.
Reformatting and cloning SMT cDNAs into the expres-
sion vector pYeDP60. Deletion of the 5'-non-coding and 3'-
non-coding regions of the SMT cDNAs was performed by PCR
amplification using specific primers.
A. thuliana cDNA 41 1 . Specific primers were designed to
introduce a BamHT restriction site immediately upstream of the
initiation codon and a XbaI site immediately downstream of the
stop codon. Direct primer: 5'-cggcggatccATG GAC TCT TTA
ACA CTC TTC-3'. Reverse primer: 5'kggctctagaTCA AGA
ACT CTC CTC CGG TGA-3'. The SMT was amplified using 25
thermal cycles (1 min 93", 2 min 56", 3 min 72") with Thermus
aquaticus ( T i q ) DNA polymerase under the standard conditions.
The PCR product was subsequently cloned into Bluescript to
give pSK 4117. pSK 4117 was linearized with XbaI, blunted
using Klenow fragment of DNA polymerase I and subsequently
digested with BamHI; the resulting DNA insert (41 18) was li-
gated into pYeDP60 containing a BamHI site at one end and a
blunted EcoRI site at the other. The resulting plasmid was called
4118-pYeDP60.
A. thaliuna cDNA 205. Specific primers were designed to
introduce a BumHI restriction site immediately upstream of the
initiation codon and a KpnI site immediately downstream of
the stop codon. Direct primer: 5'-gccgggatccATG GAC TCG
GTG GCT CTC TAC TGC ACC GC-3'. Reverse primer: 5'-
gccgggtaccTCA TTC AGA AGC TTT CTC TGG-3'. The SMT
cDNA was amplified using 25 thermal cycles (1 min 93", 2 min
56", 3 min 72") with Pyrocaccusfuriusus (Pfu) DNA polymer-
ase under the recommended conditions. The PCR product was
digested with BamHI and KpnI and inserted between the BamHI
and KpnI sites of pSK resulting in 2051-pSK. The BamHI, KpnI
insert in pSK was extracted and subcloned into BamHI, KpnI of
pYeDP60 leading to 2051-pYeDP60.
N. tabucum cDNA 132. Specific primers were designed to
introduce an EcoRV restriction site immediately upstream of the
initiation codon and a KpnI site downstream of the stop codon.
Direct primer: 5'-gccggatatcATG GAC TCT CTC ACT
CTC-3'. Reverse primer: 5'-gccgggtaccTTA CTC TTC AGG
TTT TCT GCA-3'. The SMT cDNA was amplified using 25
thermal cycles (1 min 93", 2 min 56", 3 min 72") with Pfu DNA
polymerase in the recommended conditions. pYeDP60 was lin-
earized with BamHI, blunted using Klenow fragment of DNA
polymerase I and subsequently digested with KpnI. The PCR
product was inserted between the blunted BarnHI and the KpnI
sites of pYeDP60 resulting in 1323-pYeDP60.
Nucleotide sequence determination. The nucleotide se-
quence of cDNAs 41 1 and its PCR derivative 41 18 was deter-
mined manually. The sequencing of cDNAs 412, 132, 205, rmt
and the PCR derivatives 2051 and 1323 was performed with an
automatic sequencer Perkin Elmer model 373 using T3 and T7
primers, specific oligonucleotide sequences belonging to the se-
quenced gene and a modified Taq polymerase capable of incor-
porating fluorescent dNTP. Complete sequencing of both strands
of DNA was performed. All cDNAs were in pSK except 1323
which was in pYeDP60.
Transformation of yeast. Transformation was performed
according to Schiestl and Gietz [19] with some modifications.
A fresh yeast culture (initial absorbance = 0.2) was grown in
complete medium YPG [10 g/l yeast extract (Difco), 10 g/l bac-
topeptone (Difco), 20 g/l glucose] for 5 h. The cells were col-
lected, washed twice with water and then with 1.5 ml of a 0.1 M
lithium acetate (LiAc) solution in TrisEDTA buffer (1 mM
EDTA, 10 mM Tris/HCI, pH 7.5 for transformation of erg6 and
pH 5.0 for erg2) and finally resuspended in 200 pl of the same
solution. The strain erg2 had to be sonicated 4 min before trans-
formation. Salmon sperm was added as DNA carrier (100 pg
from a 10 mg/ml solution in TrisEDTA) after sonication (10 s)
and boiling (20 min) to the plasmid DNA (1 pg). Competent
yeast cells (SO-80 pl) and 50 ml of a 40% poly(ethylene gly-
col), 0.1 M LiAc solution in TrislEDTA (pH 7.5 for erg6 and
pH 5.0 for erg2) were added. The mixture was incubated 30 min
at 30"C, then 15 min at 42°C. After centrifugation, erg6 cells
were resuspended in YPG (1 ml), incubated 1 h at 30"C, col-
lected and then plated (with 100 pl water) on minimum medium
(YNB) containing suitable supplements (histidine and adenine,
50 pg/ml each). The erg2 pellet was directly plated after the heat
shock.
Sterol analysis. Sterol isolation from lyophilized yeast cells
[20],separation of 4,4-dimethyl-, 4rx-methyl and 4-desmethyl
sterols by TLC and their acetylation [21] were performed as
described previously. TLC on silicagel plates impregnated with
AgNO, allowed to further separate the acetates, using cyclohex-
ane/toluene (6:4, by vol.) as eluent. After two migrations, ace-
tates of cycloartenol (I), obtusifoliol (111) and zymosterol (IX)
had R, of 0.45, 0.30 and 0.32, respectively. After three
migrations, acetates of Wmethylene lophenol (IV) and 24-
ethylidene lophenol (V) had R, of 0.28 and 0.50, respectively.
The acetates of stigmastadienols XVIII and XIX had the same
R, as the the acetate of zymosterol (IX). Steryl acetates were
identified by GC on a DB-1 capillary column (according to their
relative retention time to the internal standard cholesterol), then
by GC-MS and, when a sufficient amount was available, by 'H-
NMR.
GC-MS. GC-MS was performed on a computerized gas-
chromatograph mass spectrometer (Fison MD800) equipped
with an on column injector and a capillary column
(30 mX0.25 mm internal diameter) coated with DB5 (J & W
Scientific). Different fragments obtained are designed by the
ratio mlz and their relative intensity.
Sterol composition of erg6-4118-pYeDP60. GC-MS of ace-
tates of lanosterol (VI), zymosterol (IX), 5a-cholesta-7,24-dien-
38-01 (X), fecosterol (XVI), 5n-stiginasta-8,Z-24(24')-dien-3p-o1
(XVIII), d'-avenasterol (XIX), ergosterol (XX) and 5a-stig-
masta-S,7, E-22-trien-3p-01 (XXI) were described previously
[13]. 4,4,14a-Trimethyl-5a-ergosta-8,24(24')-dien-3~-yl acetate
(eburicol, XI): 482 (M') (40), 467(100), 407(94), 383(11),
323(23), 301(34), 283(18), 255(23), 241(49). 4,4,14a-trimethyl-
5rx-stigmasta-8,Z-24(24')-dien-3P-ylacetate (XII): 496 (M-)
(37), 481(100), 421(96), 383(25), 323(37), 301(20), 283(29),
255(29), 241138). 4,4-Dimethyl-5a-ergosta-8,24(24')-dien-3/?-yl
acetate (XIII): 468 (M') (loo), 453(40), 408(35), 393(58),
341(49), 283(16), 281(34), 255(56), 241(64). 4,4-dimethyl-5u-
stigmasta-8,2-24(24')-dien-3/l-y1 acetate (XIV): 482 (M-)
(loo), 467(55), 422(28), 407(61), 384(46), 341(58), 283(21),
281(32), 255(36), 241(77). 4a-Methyl-Sa-stigmasta-8,Z-24(24')-
dien-3P-yl acetate (XV): 468 (M') (94), 453(72), 408(28),
393(65), 370 (54), 355(18), 327(80), 302(14), 269(24), 267(21),
243(40), 241(53), 227(100), 225(26). MS of XI, XI11 and XV
were in good agreement with literature data ([22, 23, 211, re-
spectively). structures of XI1 and XIV were deduced from their
fragmentation pattern.
4a-Methyl steryl acetate XXX: 482 (M') (loo), 467(57),
422(31), 407(45), 355(16), 327(51), 302(9), 269(23), 267(19),
243(23), 241(64), 227(S9), 225(25).
Sterol composition of erg2-4118-pYeDP60. GC-MS of ace-
tates of lanosterol (VI), eburicol (XI), 4,4-diniethyl-5a-ergosta-
 Bouvier-NavC et al. ( E m J. Biochern. 246)
8,24(24l)-dien-3P-ol (XU), 4,4-dirnethyl-5a-stigmasta-8,2-
24(24l)-dien-3P-ol (XIV), 4a-methyl-5a-stigmasta-8,Z-24(24')-
dien-3&ol (XV), 4a-methyl sterol (XXX), 5a-ergosta-8,24(24')-
dien-3P-01 (fecosterol, XVI) and 5a-stigmasta-8,Z-24(24')-dien-
3P-01 (XVIII) were as described before [I31 or above. 5a-Ergost-
8-en-3P-yl acetate (XXII) : 442 (M') (85), 427(28), 382(7),
367(27), 315(15), 288(11), 273(18), 255(47), 229(73), 213(100).
Sa-Stigmast-8-en-3P-yl acetate (XXIII) : 456 (M') (71), 441(24),
396(7), 381(23), 315(16), 288(17), 273(14), 255(48), 229(72),
213(100). 5a-Ergosta-8,E-22-dien-3P-yl acetate (XXIV) : 440
(M') (23), 425(12), 380(5), 365(20), 315(24), 313(56), 288(38),
255(87), 241(49), 229(84), 213(52). Sa-Stigmasta-8,E-22-dien-
3P-yl acetate (XXV): 454 (M') (22), 439(11), 394(5), 379(17),
315(28), 313(55), 288(39), 255(100), 241(45), 229(95), 213(53).
Sa-Ergosta-5,8,E-22-trien-3P-yl acetate (XXVI): 438 (M+) (I),
378(60), 363(81), 337(9), 253(55), 211(32), 157(100). So-Stig-
niasta-5,8,E-22-trien-3P-yl acetate (XXVII) : 392 (M'-60) (49),
377(68), 351(5), 253(54), 211(31), 157(100). Sterols XXII to
XXVI were identified according to Rahier and Benveniste [22].
The structure of sterol XXVII was deduced from its fragmenta-
tion pattern.
'H-NMR. NMR was performed on a Bruker 400-MHz spec-
trometer. The spectra were measured in CDCI,. The chemical
shifts of signals are given in ppm with tetramethylsilane as the
internal standard, J in Hz.
Substrates for the enzymatic studies. Potential substrates
were purified by normal and AgN0,-impregnated silicagel TLC.
Extraction of germinated barley according to [24] provided 24-
methylene lophenol (purity, 95 %, according to GC). Cycloar-
ten01 (a gift of Pr. Ourisson) had the same degree of purity.
Obtusifoliol was isolated from calli of the tobacco mutant LAB
1-4 grown on LAB 170250F [25] (purity, 92%). Zymosterol
was accumulated in erg6 grown on tridemorph [26] and purified
(94%). Their MS were fully consistent with literature data ([21,
27, 28, 131, respectively). Lanosterol from Sigma was similarly
purified (99%).
Subcellular fractionation. erg6-4118-pYeDP60 cells were
disrupted as described [29] except that KCI was omitted in the
washing buffer, and BSA (1 %) added in the disruption medium.
The homogenate was centrifuged for 10 min at 12OOOXg and
the supernatant for 60 min at 1OOOOOXg. The microsomal pellet
was resuspended (5 - 10 mg proteidml) in 50 mM Tris/HCl
pH 7.5 containing 20% (by vol.) glycerol and kept at -80°C
for months without significant loss of activity. Acetone powder
of microsomes was prepared as described [30] then resuspended
and kept frozen as microsome preparations.
Enzymatic assays. A radiochemical assay was performed
with [methyl-'H]AdoMet according to Fonteneau et al. [8] but
at a smaller scale: the incubation mixture (100 pl) contained the
sterolic substrate (routinely 25 pM), Tween 80 (0.1 %), [methyl-
'HIAdoMet (475 000 cpm, usually 100 pM), protein (6- 12 pg)
and 50 mM Tris/HCI, pH 7.5, with 20% glycerol. In the blanks
for microsomal assays, microsomal proteins were omitted. In the
blanks for the kinetic studies, acetone powder was present and
the sterolic substrate was omitted. Incubations were carried out
at 30°C for 4-12 min and stopped by 100 p1 12% ethanolic
KOH. Appropriate sterol carriers were then added. The neutral
lipids were extracted with hexane and the sterols were purified
by TLC as described previously [21]. The 4,4-dimethyl-, 4a-
methyl and 4-desmethyl sterols were separately scraped off the
plate and their radioactivity determined in a liquid-scintillation
spectrometer.
A large-scale, high-yield assay was set up for GC and GC-
MS. When a high quantity of the product(s) of the enzymatic
reaction was needed, the incubation mixture (2 ml) had the same
composition as above except that unlabeled AdoMet (200 pM)
521
and a higher protein concentration (0.6 mg/ml) were used.
Longer times of incubation were also applied (Fig. 5). Sterols
were extracted and purified as in the radiochemical assay, then
acetylated and identified by GC-MS as in the sterol analysis
section. 24-Methylene cycloartanyl acetate (11) : 482 (M') (7),
467(8), 422(64), 407(66), 379(49), 300(24), 297(22), 216(32),
203(59), 201(56). 24-Ethylidene lophenyl acetate (V) : 468 (M+)
(2), 453(2), 393(2), 370(32), 355(4), 327(100), 310(5), 295(6),
267( 12), 227(12). 5a-Ergosta-7,24(24')-dien-3[j-yl (episteryl)
acetate (XVII): 440 (M') (4), 425(8), 380(3), 365(7), 356(18),
341(6), 313(100), 255(12), 253(15), 213(23). These MS were in
full agreement with literature data ([22, 24, 311, respectively).
Fecosteryl acetate (XVI), stigmasta-8,2-24(24')-dien-3D-y1ace-
tate (XVIII) and d7-avenasteryl acetate (XIX) had MS in full
agreement with those of Husselstein et al. [13].
RESULTS
Isolation and sequence analysis of SMT cDNAs
from A. thaliana, N. tabacum and R. communis
A. thaliana cDNAs. The systematic screening of an A. thali-
anu seedlings cDNA library (expressed sequence tag project)
resulted in the identification of a cDNA (VBVEC07) having
significant identity with ERG6, a yeast gene encoding a methyl-
transferase capable of converting zymosterol (IX) to fecosterol
(XVI) 132-341 (Fig. 1). Complete sequencing of this cDNA in-
dicated 38% identity with ERG6 but also showed that the cDNA
was truncated at the 5' end. A PCR fragment containing the 5'
end of the cDNA was amplified from a DNA sample of the
cDNA library using one oligonucleotide primer in antisense
orientation (PB23, 5'-GAGAAGATTCCAGTCTC-3') deduced
from the sequence of VBVEC07 and an oligonucleotide com-
plementary to T3 promoter. The cloned fragment was sequenced
and allowed us to reconstruct a full-length cDNA sequence of
1249 bp (cDNA 205) and to deduce an ORF encoding a protein
of 359 amino acids.
A probe (782 bp) was synthesized from cDNA 205 by PCR
using two oligonucleotide primers deduced from the sequence
(PB 22, 5'-ATCTACGAGTGGGGATGG-3'. and PB 23). This
PCR product was then used to screen a cDNA library of A.
thaliana siliques (400 000 recombinants) resulting in the isola-
tion of a full-length cDNA (411) of 1411 bp encoding a protein
of 361 amino acids, 38% identical with ERG6 and 82% identical
with cDNA 205.
N. tabacum cDNAs. First a cDNA probe (782 bp) was syn-
thesized by PCR using a cDNA library of tobacco (N. tabacum,
L. xanthi) calli as a template and the two oligonucleotides prim-
ers PB 22 and PB 23 deduced from putative conserved domains
of the sequence of cDNA 205. This cDNA probe was used to
screen 400000 recombinants from the tobacco calli cDNA li-
brary, resulting in the isolation of 17 cDNA clones. After se-
quencing, one of them (132) was shown to correspond to a full-
length cDNA of 1264 bp encoding a protein of 357 amino acids,
84% identical with 411. The other one (412) corresponded to a
truncated cDNA of 1276 bp encoding a protein of 352 amino
acids, 86% identical with 132. After alignment of 412 over 132,
it became apparent that 5 amino acids were lacking at the N-
terminal side of cDNA 412.
R. cominunis cDNA. First a cDNA probe (525 bp) was syn-
thesized by PCR using an EST clone (Genbank ID T23248) that
has significant identity with ERG6 as a template and two oligo-
nucleotide primers (PB 70, S-GACTTCATGAAAATGCCATT-
3' ; PB 7 1, S'-GAAGAACATTGGTGTGAAAATCTC-3') de-
duced from putative conserved domains of T 23248. This cDNA
probe was used to screen 500000 recombinants from a castor
522 Bouvier-Navt et al. (EUKJ. Biochem. 246)

2132-1 1
2412-5 1
2205-1 1
2411-2 1
Zrmt-5 1
Zsmt-2 1 -QKPEKYH
Zerg6 1
2132-1 53
2412-5 47
2205-1 52
2411-2 52
Zrmt-5 34
Zsmt-2 55
Zerg6 48
2132-1 115 AVDLIGVKPGARI
2412-5 110 AVDLLGIKPGARV VNRARAHNKKAGLDSQCEV
2205-1 115 AVDLIKVKPGQKI VQRAKLHNKKAGLDSLCNV
2411-2 115 AVDLIQVKPG-KI VNRARLHNKKAGLDALCEV
Zrmt-5 92 LALQLGLKPEQKV ITRGKVLNRIAGVDKTCDF
Zsmt-2 113 ITRGKELRNIAGVDKTCNF
Zerg6 111
2132-1 178 ELYRPEDP
2412-5 173 ELYNSDDP
2205-1 178 EKYRDDDE
2411-2 177 EKFKAEDD
Zrmt-5 155 DSFDPNNQ
Zsmt-2 176 DSFDPQNP
Zerg6 174 DKPDENNP
2132-1 241 VEIIHG
2412-5 236 VKIIHG
2205-1 241
2411-2 240
Zrmt-5 218
Zsmt-2 239
Zerg6 237

132-1 291 -----TRLKMGRIAPWRNHILVTILAFL HM

412-5 286 -----TRLKMGRIAPWRNHIVVTVLSWL HM
205-1 291 - - - - -NRLKMGRIAPWRNHVVVVILSAI nu
411-2
rmt-5
290
274
-----TRLKMGRLAYWRNHIVVQILSAV
Q P SL T G - F R L T A IG R F F T R N M IK A L B F A
HM
FF
smt-2 295 H F SL S S - F R L T A V O R L F T K N M V K V L E Y V PF
erg6 300 LANLATFFRTSYLGRQFTTAMVTVUEKL ML
2132-1 349 ILCR BE-------------
2412-5 344 ILCR EEH------------
2205-1 349 ILCR EKASE----------
2411-2 348 ILCR ESPEESS--------
Zrmt-5 336 FLAQ HSENQ----------
Zsmt-2 357 FLAR DLDRN----------
Zerg6 363 FVAR ENAETPSQTSQEATQ

Fig. 2. Sequence alignment of the sterol methyltransferases. Alignment was performed using the PILEUP program of the UCCG package
run with the default parameters Positions with a consensus residue present in the seven sequences are boxed. 2132-1 and 2412-5 stand for two
SMT found in N tubacum (this work) 2205-2 and 2411-2 stand for two SMT found in A. thalianu (this work) Zrmt-5, R. communis SMT (this
work). Zsmt-2, G. mux SMT [12]. Zerg6, S. cerevzszue SMT [33].The sequences considered in the discussion are underlined.

bean cDNA library, resulting in the isolation of 27 cDNA clones.
After sequencing one of them (rmt) was shown to correspond to
a full-length cDNA of 1328 bp encoding a protein of 346 amino
acids, 39% identical with 411, 51% identical with the protein
encoded by ERG6.
Comparison of deduced aminoacid sequences. The five
above-mentioned amino acid sequences were aligned using the
Pile-up program and compared with the amino acid sequence
deduced from the G. m a cDNA encoding an AdoMet: AZ4-ste-
rol-C-methyltransferase [ 121 and the amino acid sequence of
ERG6 (Fig. 2).
This comparison reveals two main features. These sequences
present highly homologous regions : one of them (IN)LD(A/V)-
GCG(V/I)GGP corresponds to the consensus motif described by
several authors [35-371 for all AdoMet-dependent 0-,N- and
C-methyltransferases catalyzing methyl transfer on e.g. caffeic
acid M73235 [38], phosphatidyl ethanolamine LO7247 and di-
hydroxypolypreny lbenzoate L20427 [391, respectively. A second
one is an invariant motif IEATCHAP not present in other meth-
yltransferases and possibly typical of methyltransferases acting
on a sterol substrate. Moreover, these seven amino acid se-
quences can be divided in at least two groups: the first one
contains the G. max [I21 and the R. communis methyl-
transferases, the second one contains the four methyltransferases
from A . thaliana and N. tabacum (205, 411, 132, 412). SMT
from the second group are more than 80% identical in all pos-
sible combinations but are less than 40% identical with SMT
from the first group. In this first group R. communis SMT is
83 % identical with G. max SMT. Whereas SMT from the second
group possess a hydrophobic domain of approximately 25 amino
acids at the N-terminal position, G. max and R. communis SMT
are devoid of such a hydrophobic domain. The yeast SMT
encoded by ERG6 is 50% and 38 % identical with plant SMT of
the first and second group, respectively. In addition, ERG6 SMT
has no hydrophobic domain at the N-terminal position. There-
fore ERG6 is closer to the first group than to the second.
 Bouvier-NavC et al. (Eul: J. Biochenz. 246) 523
Table 1. Sterol composition of mutant yeast strains erg6 and erg2 transformed with plasmid pYeDP60 with or without ORF 4118. Results
are given as percentages of the total sterol content. Sterols were identified by their RRT in GC and fragmentation pattern in GC-MS.

Sterol class Sterol compound Composition of

ergb- erg6-4118- erg2- erg2-4118-

pYeDP60 pYeDP60 pYeDP60 pYeDP60

% of total

4,4-dimethyl sterols lanosterol" (VI) 7.5 6

eburicol a (XI) 12.5 3.5
(XII)
4,4,1Ja-trimethyl-5a-stigmasta-8,Z-24(24')-dien-3~-ol 3 2.5
(XIII)
4,4-dimethyl-5a-ergosta-8,24(24')-dien-3P-o1 2 1
(XIV)
4,4-dimethyl-5a-stigmasta-8,2-24(24')-dien-3P-o1 6 6.5
4a-methyl sterols (XV)
4-methyl-5a-stigmasta-8,Z-24(24')-dien-3~-oI 6 5
xxx 0.5 1
4-desmethyl sterols zymosterol" (IX) -
19.5
5n-cholesta-7,24-dien-3~-ol (X) 2 -

5a-stigmasta-8,Z-24(2J1)-dien-3P-ol(XVIII) 22 34
A'-avenasterol* (XIX) 11 -

ergosterol" (XX) 6 -

(XXI)
5a-stigrnasta-5,7,E-22-trien-3P-o1 2 -

fecosterol (XVI) - 2
5a-ergosta-8-en-38-01 (XXII) - 23
5a-ergosta-S,E-22-dien-3,8-01 (XXIV) - 4.5
5a-ergosta-5,8,E-22-trien-3P-o1(XXVI) - 2
5a-stigmasta-8-ene-3P-01 (XXIII) - 4.5
5a-stigmasta-8,E-22-dien-3P-ol (XXV) - 3
5a-stigrnasta-5,8&22-trien-3P-o1 (XXVII) - 0.5

(XI) : zymo-
Lanosterol = 4,4,14a-trirnethyl-5n-cholesta-8,24-dien-3,!-01(VI); eburicol = 4,4,14a-trimethyl-5a-ergosta-8,24(24')-dien-3P-ol
sterol 1 5a-cholesta-8,24-dien-3P-o1 (IX); fecosterol = Sa-egosta-8,24(24')-dien-38-ol (XVI); A7-avenasteroI = 5a-stigmasta-7,Z-24(24')-dien-
3P-01 (XIX) : ergosterol = Sa-ergosta-5,7,E-22-trien-3P-o1(XX).

Expression of A. thulianu SMT cDNA 411 in erg6. The sterolic
composition of erg6-4118-pYeDP60, first described in Hus-
selstein et al. [13], was determined at higher scale in order to
detect minor compounds and to further characterize the major
compounds. In addition to the 4-desmethyl sterols IX, X, XVIII,
XIX, XX and XXI [I31 several precursors were identified by
GC and GC-MS (Table 1 and Fig. 3).
Sterols XI and XI1 are the 24-methylene and 24-ethylidene
derivatives, respectively, of lanosterol (VI). Sterols XI11 and
XIV are the same derivatives for 4,4-dimethylcholesta-8,24-di-
enol (VII) and sterol XV is the 24-ethylidene derivative of 4a-
methyl-cholesta-8,24-dienol (VIII).
Another 4a-methyl sterol (XXX) was detected, the GC-MS
of which corresponds to the skeleton of XV with an additional
-CH,- in the side chain. The MS does not allow us, however, to
localize in the side chain the extra methylene; sterol XXX might
bear, on C24, an isopropylidene, isopropenyl or propenyl group.
Although stigmasta-8,24(24')-dienol (XVIII) is the major
sterol of erg6-4118-pYeDP60, it could not be easily isolated
from this strain for NMR analysis because of the presence of its
A7-isomer (XIX) and zymosterol (IX) which migrated with
XVIII during TLC on AgN0,-impregnated silicagel. The trans-
formation of the yeast mutant erg2 was performed for this pur-
pose.
Expression of SMT cDNAs from A. thuliunu (205) and N.
tubacum (132) in erg6. As mentioned in the introduction, it has
been postulated that the two methylation reactions occurring in
higher plant synthesis are catalyzed by two different enzymes
[9] (Fig. 1). The coexistence of two putative SMT cDNAs pre-
senting about 80% identity in either A. thaliana or N. tabacum
suggested they might encode each of these two enzymes. To
check this hypothesis these cDNAs were expressed in the null
mutant erg6. To be able to compare reliably results of expression
experiments, cDNAs 411, 205 and 132 were formatted iden-
tically and inserted in pYeDP60 (see Experimental Procedures)
resulting in erg6-205 1-pYeDP60 and erg6-1323-pYeDP60 in ad-
dition to the above erg6-4118-pYeDP60.
The sterolic composition of erg6-2051-pYeDP60 and ergh-
1323-pYeDP60 was close to that of erg6-4118-pYeDP60 (data
not shown). The main feature was the de novo synthesis of 5a-
stigmasta-8,2-24(24')-dien-3P-o1 (XVIII) and d'-avenasterol
(XIX) which represent in the three cases more than 30% of total
sterols. These results strongly support the idea that A. thaliana
cDNAs 41 1 and 205 and N. tubacum cDNA 132 encode catalyti-
cally identical enzymes which would all be involved in the sec-
ond methylation step of sterol biosynthesis (Fig. 1).
Expression of A. thaliuna SMT cDNA 411 in erg2. To accumu-
late stigmasta-8,24(24')-dienol (XVIII) in the absence of its 4'-
isomer (XIX), we transformed the yeast mutant erg2 which lacks
Ax--d7-~tero1 isomerase [40]. The sterol composition of erg2-
4118-pYeDP60 is shown in Table 1, together with that of erg2
transformed with the void plasmid. Sterol XVIII was also the
major sterol in erg2-4118-pYeDP60 and neither sterol XIX nor
zymosterol (IX) were detected, thus allowing the purification of
XVIII and its clear identification as 5a-stigmasta-8,2-24(24')-
dien-3P-01 by the 'H NMR spectrum of its acetate: 6 0.613
(3H,s, Hlg), 0.962 (3H,d, J = 6.4, H21), 0.965 (3H,s, H19),
0.978 (6H,d, J = 6.8, H26 and 27), 1.590 (3H,d, J = 6.8, H29),
2.829 (lH,septet, J = 6.9, H25), 4.702 (1H,m, H3a), 5.109
(lH,quartet, J = 6.9, H24'), in full agreement with data of
Schmitt and Benveniste [21].
The sterol composition of erg2 transformed with the void
plasmid is similar to that described for the first isolated erg2
strain [41]. It contains mostly 4-desmethyl sterols: XVI, XXII,
524 Bouvier-NavC et al. (Eul: J . Biochem. 246)

ASMT

XI1

-
A.SMT

xn1 XIV

ASMT ASMT
-

xv

...,,&
A.SMT
- L / )
ASMT

H XVI XVIII

i
i
A8-A7-sterolisomerase
I A8-A7-sterol isomerase
i
I

-
'

- '1
&-
'% ,%&

A.SMT A.SMT

HO XVII XM

#'I!: &'1 1 C-5 desaturase

A24(24l) reductase
C1-22 desaturase
-/

H \
xx Ho XXI
Fig. 3. Proposed sterol biosynthesis pathways in the yeast mutant erg6 (dotted arrows) and in erg6 transformed with ORF 4118 in pYeDP60
(dotted plus solid arrows). Sterols VI, IX and X were present in both erg6-pYeDP60 and erg6-4118-pYeDP60 (Table 1). Sterols XI-XXI were
found in erg6-4118-pYeDP60(Table 1) with the exception of sterols XVI and XVII which appeared after incubation of zymosterol in vitro (Fig. 5 ) .
A. SMT = A. thaliana SMT.

XXIV and XXVI (Fig. 4). In the 4-desmethyl sterols fraction
of erg2-4118-pYeDP60, in addition to these four sterols of the
ergosta-series, the counterparts of the stigmastaseries are ob-
served, i.e. sterols XVIII, XXIII, XXV and XXVII, respectively
(Table 1 and Fig. 4).
The same 4,4-dimethyl sterols (XI, XII, XIII, XIV) and 4a-
methyl sterols (XV and XXX) appeared in erg2 as in erg6 when
they were transformed with the A. thaliana SMT. Thus, in two
different yeast strains transformed with ORF-4118, it was shown
that A. thulium SMT (a) can accept four different tetracyclic
skeletons, 4,4,14-trimethyl, 4,4-dimethyl, 4a-methyl or 4-
desmethyl sterol, and (b) can methylate either a sterol 424(25)
or 424(24') double bond. The detection of sterol XXX in both
transformed strains further suggests that the A. thaliana enzyme
could catalyze, in certain circumstances, a third methylation of
the side chain.
Characterization and substrate specificity of A. thaliana
SMT from erg6-4118-pYeDP60. In preliminary experiments,
microsomal preparations from erg6-4118-pYeDP60 were incu-
bated with [n~ethyl-~HIAdoMet in the absence of exogenous ste-
rol substrate. After extraction and purification by TLC, sterols
were found to be significantly labelled, indicating that micro-
somes from erg6-4118-pYeDP60 do contain a SMT activity and
that the enzyme uses as substrates the endogenous sterols associ-
ated with the microsomal membranes.
In contrast, microsomal preparations from erg6 transformed
with the void plasmid (erg6-pYeDP60) were devoid of SMT ac-
 Bouvier-NavC et al. (Eur J. Biochem. 246) 525

.......SMT
erg2
-
A.SMT
.....
m
a+
,.,&
- - .,.,rJs
A.SMT
,
% ,
.,&

HO XVI XVIII

i
A24(24')reductase
I
LI) LI)

XXII xxm

XXIV XXV

I
&&
i C-5-desaturase
i

HO HO

XXVI XXVII
Fig. 4. Proposed sterol biosynthesis pathways, downstream from zymosterol, in the yeast mutant erg2 (dotted arrows) and in erg2-4118-
pYeDP60 (dotted plus solid arrows). Zymosterol (IX) was found in the biochemical analysis of erg2 transformed with pYeDP60. The biosynthesis
pathways upstream from zymosterol are identical to those in erg6 and erg6-4118-pYeDP60,respectively (Fig. 3).

tivity. Indeed no radioactivity was incorporated in the sterols
after incubation with labelled AdoMet, in agreement with the
absence of endogenous SMT in the yeast null mutant erg6.
The significant methylation of sterols present in the micro-
soma1 membranes of erg6-4118-pYeDP60 upon incubation with
[methyl-'Hl AdoMet prevented the accurate determination of the
methylation of exogenous sterol substrates. To eliminate these
endogenous sterols, the microsomes of erg6-4118-pYeDP60
were delipidated with cold acetone. When the resulting acetone
powder was incubated with labelled AdoMet, no significant ra-
dioactivity was incorporated in sterols if no exogenous sterols
were added; the specific activity of SMT in acetone powder
preparations, measured with 24-methylene lophenol, was as high
as that of microsomes. The acetone powder was thus used in all
further studies.
Conditions under which A. thaliana SMT activity of acetone
powder from erg6-4118-pYeDP60 was proportional to time and
protein concentration were set up in the presence of either cyclo-
artenol (I) or 24-methylene lophenol (IV), the respective sub-
strates of the first and the second methylation steps in plants
(Fig. 1). The kinetic parameters of the SMT activity of erg6-
41 18-pYeDP60 towards various potential sterolic substrates
were then determined using the radiochemical assay (Table 2).
Cycloartenol (I) and 24-methylene lophenol (IV), were first
compared. While the K,, for IV (5 pM) was half that for I, the
V,,, determined with IV (around 280 nmol . mg protein-' .
h-I) was about seven-times higher than that measured with I.
Hence the catalytic efficiency of the A. thaliana SMT, indicated
by the ratio V,,,IK,,, is about 17-times higher with 24-methylene
lophenol (IV) than with cycloartenol (I).
Lanosterol (VI) and obtusifoliol (111) were then compared
with cycloartenol (I) as substrates for the A . thaliana SMT. La-
nosterol (VI) had kinetic parameters similar to cycloartenol (I)
whereas obtusifoliol (111) displayed a similar K,, but a V,,, value
about half that of I.
The sterol composition of erg6-4118-pYeDP60 (Table 1)
clearly suggested that zymosterol (IX), the major sterol of the
mutant erg6, is significantly methylated by the A. thaliana SMT
since products XVIII to XXI accumulated. In vitro, zymosterol
also proved to be a good substrate of A. thaliana SMT since its
efficiency of methylation (V,,,,,IK,,,) was 71 % that of 24-methy-
lene lophenol (Table 2).
Identification of the methylation product(s) of 24-methylene
lophenol, cycloartenol and zymosterol by the A. thaliana
SMT. Under conditions where enough product was formed to
allow GC and GC-MS analysis, we could clearly confirm that
24-methylene lophenol (IV) was transformed in 24-ethylidene
lophenol (V) and cycloartenol (I) in 24-methylene cycloartanol
(11) (Fig. 5). Zymosterol (IX) gave rise to 4 products: fecosterol
(XVI), episterol (XVII), stigmasta-8, 24(24')-dienol (XVIII) and
d7-avenasterol (XIX). This result, clearly confirms that A. thali-
ana SMT can perform two successive methylations, allows us
to complete the sterol biosynthesis scheme shown in Fig. 3 since
526 Bouvier-Navt et al. (Eul: J, Biochem. 246)
A I
I
Fig. 5. Gas chromatograms of sterols I, IV and IX before (t = 0) or
after incubation with AdoMet and the acetone powder from ergb-
4118-pYeDP60. (A) Cycloartenol I, (B) 24-methylene lophenol IV, and
(C) zyinosterol IX were incubated for various times with AdoMet under
the large-scale, high-yield conditions (see Experimental Procedures).
Relative retention times (RRT) of steryl acetates on DB-1 column with
cholesterol as internal standard (RRT = 1) were, cycloartenol (I), 1.46;
24-methylene cycloartanol (II), 1.53 ; 24-methylene lophenol (IV), 1.41;
24-ethylidene lophenol (V), 1.56; zymosterol (IX), 1.23; fecosterol
(XVI), 1.31; episterol (XVII), 1.35 ; stigmasta-8,2-24(24')-dienol
(XVIII), 1.46 and A'-avenasterol (XIX) 1.50.
fecosterol (XVI) and episterol (XVII) were not detected in the
biochemical analysis of erg6-4118-pYeDP60 (Table l), and indi-
cates that the endogenous AX-A7-isomerasefrom erg6 is present
and active in the acetone powder of microsomes from e r g 6
4118-pYeDP60. The presence of A8-d7-stero1isomerase activity
was confirmed by (a) the partial conversion of zymosterol (IX)
to cholest-7,24-dienol (X) when incubated in the absence of
AdoMet and (b) the conversion of stigmasta-8,24(24')-dienol
(XVIII) to A'-avenasterol (XIX) under similar experimental con-
ditions (data not shown).
DISCUSSION
Sequence comparisons of polypeptides deduced from SMT
cDNAs. In the present study we report the isolation of five
cDNA sequences encoding plant AdoMet-dependent SMT. This
conclusion is based on the identity existing between these se-
quences and ERG6, a gene from yeast encoding a zymosterol
C24-methyltransferase [32-341. In addition database searching
revealed similarities between the five polypeptides encoded by
these cDNAs and several other known methyltransferases. In a
thorough study Kagan and Clarke [35] have reported three con-
sensus motifs present in most methyltransferases and probably
involved in the binding of AdoMet. The first motif, remarkably
Table 2. Comparison of the apparent kinetic parameters for various
potential sterolic substrates of the A. thaliana sterol methyl-
transferase expressed in erg 6. The kinetic parameters were determined
after incubation of 100 pM [methyl-'H]AdoMet with (i) IV or IX (2-
20 pM) and 0.06 mg proteiniml (acetonic powder from erg6-4118-
pYeDP60 microsomesj for 4 min, or (iij I, VI or I11 (4-40 pM) and
0.12 mg protein/ml for 13 min. Under these conditions, the conversion
yield of the substrates never exceeded 10%. With all the tested sterols,
saturation kinetics followed the Michaelis-Menten equation. However,
since the assay medium is an heterogeneous mixture (soluble AdoMet
+ detergent-emulsified sterols + resuspended proteins) the determined
K,,, and V,,,, values are termed apparent.
% relative to pM % relative to
24 -Meth y1ene 24-Methylene
lophenol lophenol
24-Methylene lophenol IV" 100 4.9 100
Cycloartenol I 14 11.4 6
Lanosterol VT 13 12.4 5
Obtusifoliol 111' 7 11.9 3
Zymosterol IX' 75 5.2 71
* For IV, V,,,,, and K , values are means of five separate deter-
minations. Standard deviations were 22% for V,,,, (mean value =
280 nmol . mg protein-' h-') and 32% for K,.
For I, V,,,, and K,, values are means of three separate deter-
minations, with IV as reference. Standard deviations were 21 % for
V,,,,, (expressed as% V,,,,,,") and 25% for K,,,.
For VI, 111and IX, V,,,,, and K,mvalues are means of two separate
determinations, with I or IV as reference. Deviations from the means
were less than 20%.
conserved over nearly 85 methyltransferase genes, corresponds
to the LD(A/V)GCG(V/I)GGP domain in the five SMT polypep-
tides (Fig. 2). The second and third motifs, corresponding to
NSFDGAYS and VLKPGSMYVSY, respectively, in A. thulium
cDNA 41 1 are less conserved throughout methyltransferases
than motif I. After alignment of the deduced sequences of the
five SMT cDNAs cloned in this work plus those of G. rnux
cDNA 1121 and yeast ERG6 we observed that the seven se-
quences have common features : the AdoMet-binding motif
no. I, a typical domain YE(YIF/W)GWGXSFHF and a totally
conserved motif IEATCHAP. It is tempting to speculate that
these last two invariant domains may be involved in sterol sub-
strate binding and(or) enzymatic catalysis. However the seven
cDNAs also present important differences allowing us to divide
them into at least two groups. The first group includes the G.
mux and R. communis cDNAs which are 83% identical to each
other. The second group corresponds to the two A. thulium (411,
205) and N. tabucum (205, 412) cDNAs which are more than
80% identical in all combinations but are less than 40% iden-
tical with members of the first group. This second group pos-
sesses a stretch of about 20-25 hydrophobic amino acids which
could correspond to a transmembrane domain involved in the
association of these SMT with the endoplasmic reticulum 1421.
This hydrophobic domain is not present either in the first group
of cDNAs or in ERG6 which in many aspects is closer to the
first group.
Functional expression of SMT cDNAs in erg6. The A. thulium
cDNAs 411 and 205 and the N. tabucum cDNA 132 were ex-
pressed in the yeast null mutant erg6. Being deficient in AdoMet
zymosterol C24-methyltransferase, this mutant is devoid of 24-
alkylated sterols (Table 1). Its transformation by any of the three
cDNAs resulted in a sterol profile where stigrnasta-8,24(24')-
dienol (XVIII) and d'-avenasterol (XIX) were the major sterols.
 Bouvier-NavC et al. (Eul: J. Biochem. 246)
In other words, all three cDNAs encode an enzyme which can
produce in vivo 24-ethylidene sterols from 24-non-alkylated
sterols.
The sterol composition of erg6-4118-pYeDP60 was thor-
oughly studied by GC-MS. In addition to the previously de-
scribed 4-desmethylsterols IX, X, XVIII to XXI [13j, the inter-
mediate sterols XI to XV were identified (Fig. 3). Sterols XI
and XI1 correspond to the products of the first and the second
methylation, respectively, of lanosterol (VI). Although we did
not detect sterols VII and VIII in either erg6-pYeDP60 or e r g b
41 18-pYeDP60, they are known intermediates of yeast sterol
biosynthesis [43, 441 and hence they are the rational precursors
of XI11 and XIV on the one hand, and XV on the other, respec-
tively. These results complement our previous study [13] and
clearly show that the A. thatiana SMT encoded by cDNA 411
is able, to recognize four different tetracyclic skeletons, 4,4,14-
trimethyl, 4,4-dimethyl, 4a-methyl or 4-desmethyl sterol, and to
methylate either a 424(25) or a 424(24') double bond.
The yeast null mutant erg2, deficient in ~ 8 - ~ 7 - ~ t eisom-
ro1
erase, was similarly transformed by 4118-pYeDP60 and its sterol
composition (Table 1, Fig. 4) totally confirmed the above con-
clusion. Since erg2-4118-pYeDP60 was devoid of zymosterol
and 4'-avenasterol, we could easily purify sterol XVIII,
the major sterol in both erg6 and erg2 transformed with Arabi-
dopsis cDNA 411. Its 'H-NMR spectrum clearly confirmed its
identification as 5a-stigma~ta-8,Z-24(24~)-dien-3P-01.
The in vitro study of A. thaliana SMT using acetone powders
of microsomes from erg6-4118-pYeDP60 was fully consistent
with the data obtained in vivo: lanosterol and zymosterol were
shown to be substrates when [3H]AdoMet was added (Table 2),
and large-scale, high-yield incubation of zymosterol allowed the
identification of its methylation products : fecosterol (XVI) and
stigmasta-8,Z-24 (24')-dien-3P-ol (XVIII) (accompanied by their
4' counterparts XVII and XIX) (Fig. 5 ) .
The aim of the in vitro study was to answer the following
question: does cDNA 411 encode a cycloartenol methyl-
transferase, (SMT, in Fig. l), a 24-methylene lophenol methyl-
transferase (SMT,) or a single SMT able to perform both reac-
tions ? When cycloartenol (I) and 24-methylene lophenol (IV)
were incubated with AdoMet and the acetone powder of micro-
somes from erg6-4118-pYeDP60 under the large-scale, high-
yield conditions, both sterols were shown to be substrates and
their methylation products, 24-methylene cycloartanol (11) and
24-ethylidene lophenol (V), respectively, were clearly identified
by GC-MS. The kinetic parameters of the reaction for these two
substrates were determined using labelled AdoMet (Table 2).
The V,, lIVmun 1v ratio was 141100. The K,, measured for I was
twice that for IV. Therefore the catalytic efficiency of the en-
zyme encoded by cDNA 411 was 17-times higher for 24-methy-
lene lophenol (IV) than for cycloartenol (I).
Previous studies on substrate specificity of plant SMT using
microsomes from bramble cells [8, 91 clearly showed that both
sterols are methylated. In addition, when cycloartenol (I) and
lanosterol (VI) were compared as substrates for methylation with
cell-free preparations from different plants or algae, I was con-
sistently a better substrate than VI. It was methylated 5-10-
times [S, 8 , 9, 451 or at least 3-times [7] more efficiently than
VI. The same comparison between I and VI as substrates for the
erg6-4118-pYeDP60 SMT showed that both sterols had equiva-
lent Vmaxsand K,s. This discrepancy between the results ob-
tained with plant enzymatic preparations, which contain either
two different SMT or an aspecific one, and the enzymatic prod-
uct of A. thaliana cDNA 411 strongly suggests that plants do
not contain a unique SMT and that the A. thaliana SMT under
study is a 24-methylene lophenol methyltransferase.
527
Considering the kinetic parameters determined with the dif-
ferent plant or yeast sterols for methylation by the A. thaliana
SMT encoded by cDNA 411 (Table 2), it is clear that the cata-
lytic efficiency of this enzyme depends more on the sterolic
skeleton than on the position 424(25) or 424(24') of the double
bond in the side chain. Thus zymosterol (IX) is methylated 70%
as efficiently as 24-methylene lophenol although it possess a
A24 double bond instead of a A24(24') double bond. Hence the
poor methylation efficiency observed with cycloartenol (I) and
lanosterol (VI) would not be due to their A24 double bond but
to their extra 4b andor 14a-methyl groups. Since obtusifoliol
(111) which is devoid of 4P-methyl group, is also a poor sub-
strate, it is likely that the common feature which hinders the
methylation of sterols I, VI and I11 is the presence of the 14a-
methyl group. Previous observations with bramble cell micro-
somes suggested the same conclusion [S, 91.
Finally, since the protein expressed by erg6-4118-pYeDP60
shows selectivity towards 24-methylene lophenol in vitro and
produces in vivo the accumulation of 24-ethyl sterols in both
erg6-4118-pYeDP60 and erg2-4118-pYeDP60, we can conclude
that the cDNA 41 1 encodes a SMT involved in the second meth-
ylation step, that is the conversion of methylene-24 lophenol
into 24-ethylidene lophenol. For reasons developed above, such
a conclusion could be extrapolated to the A. thaliana cDNA 205
and the N. tabacum cDNA 132, since these cDNAs are more
than 80% identical to 411 and null mutant erg6 transformed
with plasmids 2051 -pYeDP60 and 1323-pYeDP60 became able
to synthetize as much 24-ethyl sterols as erg6-4118-pYe-DP60.
Therefore the cDNAs of the second group encode a 24-methy-
lene lophenol C24'-methyItransferase. In contrast we suggest
that the first group of cDNAs (R. communis, G. max) could en-
code a protein involved in the first methyl transfer (C24 methyl-
ation), that is to say catalyzing the conversion of cycloartenol
into 24-methylene cycloartanol (Fig. 1). This hypothesis rests on
the finding that the first group of cDNAs is more similar to
ERG6 than the second group (see above) and that the polypep-
tide encoded by ERG6 is involved in the conversion of zymo-
sterol (IX) to fecosterol (XVI), a C24 methylation ; moreover
ERG6 polypeptide seems unable to achieve a C24' methylation,
since two 24-methylene sterols, 24-methylene cholesterol and
fecosterol (XIV) were shown not to be substrates [46j. In this
context it should be recalled that the G. max cDNA has been
expressed in E. coli as a fusion protein which was shown to
catalyze the conversion of lanosterol (VI) to eburicol (XI) and
therefore to catalyze a C24 methylation [14]. However, such a
result does not constitute a strong argument in favour of our
hypothesis since there are no comparative measurements allow-
ing to show the existence of a specificity for C24 methylation
rather than for C24l methylation, and lanosterol is not a physio-
logical substrate in higher plants, which always contain cyclo-
artenol (for review see [47]). Expression studies and measure-
ment of enzymatic activities with appropriate substrates will be
necessary to clarify this point.
We believe that the cloning of a 24-methylene lophenol
C24'-methyltransferase catalyzing the second methylation step
during plant sterol biosynthesis opens new avenues, in perform-
ing molecular enzymological studies to understand the catalyti-
cal mechanism of this fascinating enzyme [6], and in unravelling
the intricate mechanism which controls the alkylation level in
higher plant sterols. This point is important since plant mem-
branes contain a mixture of C,,, C,, and C,, sterols differing by
their methylation extent at C24 and in a defined proportion
which is inheritable in a given genotype.
We are grateful to Dr M. Bard (Indiana University-Purdue Univer-
sity Indianapolis) for kindly providing the yeast null mutants erg6 and
528 Bouvier-NavC et al. (Eul: J. Biochem. 246)
erg2 and to Dr C. Somerville (Carnegie Institute of Washington, Staii-
ford) for the EST clone no. 52309 (GenBank ID, T23248) and for a
cDNA library from R. communis. We thank also Dr D. Pompon [Centre
National de la Recherche Scientfique (CNRS), Gif sur Yvette] for allow-
ing us to use plasmid pYeDP60, Dr J. Giraudat (CNRS, Gif-sur-Yvette)
for giving us a cDNA library of A. thalianu siliques and Profs T. J. Bach
and M. Rohmer (CNRS, Strasbourg) for carefully reading the manu-
script. We warmly acknowledge the skillful assistance of P. Hamann for
the cDNA sequencing, R. Meens for the GC-MS and B. Bastian who
patiently typed the manuscript.
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