Identification of Cdnas Encoding Sterol Methyl-Transferases involved in the Second Methylation Step of Plant Sterol Biosynthesis

Eur. J. Biochem. 246, 518-529 (1997) 0 FEBS 1997

Identification of cDNAs encoding sterol methyl-transferases involved in the second methylation step of plant sterol biosynthesis Pierrette BOUVIER-NAVE’, Tania HUSSELSTEIN ’, Thierry DESPREZ2 and 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 40% 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 containing 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-6-42 18-pYeDP60 were shown to possess AdoMet-dependent sterol-C-methyltransferase activity. Delipidated preparations of these microsomes converted cycloartenol into 24-methylene cycloartanol and 24-methylene lophenol into 24-ethylidene lophenol, thus allowing the first identification 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-methionine (AdoMet) sterol C-methyltransferases (SMT). In Saccharo- myces cerevihiue, the SMT converts zymosterol (IX) into fecosterol (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 substrate 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 second 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 % identity 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

11

V

11 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 isolated from Arabidopsis thaliana in our laboratory [13]; its sequence 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-methyltransferase (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 substrate-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-methyltransferase.

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 [ lo 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 expression 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 iq) 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 polymerase 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 linearized 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 sequence of cDNAs 41 1 and its PCR derivative 41 18 was determined 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 sequenced 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 bactopeptone (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 transformation. 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, acetates 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 acetates 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-stigmasta-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-yl acetate (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, respectively). 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 acetates of lanosterol (VI), eburicol (XI), 4,4-diniethyl-5a-ergosta-

Bouvier-NavC et al. ( E m J. Biochern. 246) 521

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 fragmentation pattern.

'H-NMR. NMR was performed on a Bruker 400-MHz spectrometer. 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)

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-y1 acetate (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. thalianu 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 methyltransferase capable of converting zymosterol (IX) to fecosterol (XVI) 132-341 (Fig. 1). Complete sequencing of this cDNA indicated 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 isolation 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 synthesized by PCR using a cDNA library of tobacco (N. tabacum, L. xanthi) calli as a template and the two oligonucleotides primers 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 l i - brary, resulting in the isolation of 17 cDNA clones. After sequencing, 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 synthesized by PCR using an EST clone (Genbank ID T23248) that has significant identity with ERG6 as a template and two oligonucleotide primers (PB 70, S-GACTTCATGAAAATGCCATT- 3' ; PB 7 1, S'-GAAGAACATTGGTGTGAAAATCTC-3') deduced from putative conserved domains of T 23248. This cDNA probe was used to screen 500000 recombinants from a castor

522 Bouvier-Navt et al. (EUK J. Biochem. 246)

2132-1 1 2412-5 1 2205-1 1 2411-2 1 Zrmt-5 1 Zsmt-2 1 Zerg6 1

2132-1 53 2412-5 47

2411-2 52 2205-1 52

Zrmt-5 3 4 Zsmt-2 55 Zerg6 4 8

2132-1 115 2412-5 110 2205-1 115 2411-2 115 Zrmt-5 92 Zsmt-2 113 Zerg6 111

2132-1 178 2412-5 173 2205-1 178 2411-2 177 Zrmt-5 155 Zsmt-2 176 Zerg6 174

2132-1 241

2205-1 241 2412-5 236

2411-2 240 Zrmt-5 218 Zsmt-2 239 Zerg6 237

- Q K P E K Y H

A V D L I G V K P G A R I A V D L L G I K P G A R V V N R A R A H N K K A G L D S Q C E V A V D L I K V K P G Q K I V Q R A K L H N K K A G L D S L C N V A V D L I Q V K P G - K I V N R A R L H N K K A G L D A L C E V L A L Q L G L K P E Q K V I T R G K V L N R I A G V D K T C D F

I T R G K E L R N I A G V D K T C N F

E L Y R P E D P E L Y N S D D P E K Y R D D D E E K F K A E D D D S F D P N N Q D S F D P Q N P D K P D E N N P

V E I I H G V K I I H G

132-1 291 - - - - - T R L K M G R I A P W R N H I L V T I L A F L H M 412-5 286 - - - - - T R L K M G R I A P W R N H I V V T V L S W L H M 205-1 291 - - - - - N R L K M G R I A P W R N H V V V V I L S A I n u 411-2 290 - - - - - T R L K M G R L A Y W R N H I V V Q I L S A V H M rmt-5 274 F F smt-2 295 P F erg6 300 L A N L A T F F R T S Y L G R Q F T T A M V T V U E K L M L

Q P S L T G - F R L T A I G R F F T R N M I K A L B F A H F S L S S - F R L T A V O R L F T K N M V K V L E Y V

2132-1 349 I L C R B E - - - - - - - - - - - - - 2412-5 344 I L C R E E H - - - - - - - - - - - - 2205-1 349 I L C R E K A S E - - - - - - - - - - 2411-2 348 I L C R E S P E E S S - - - - - - - - Zrmt-5 336 F L A Q H S E N Q - - - - - - - - - - Zsmt-2 357 F L A R D L D R N - - - - - - - - - - Zerg6 363 F V A R E N A E T P S Q T S Q E A T Q

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-sterol-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 methyltransferases and possibly typical of methyltransferases acting on a sterol substrate. Moreover, these seven amino acid sequences can be divided in at least two groups: the first one contains the G. max [I21 and the R. communis methyltransferases, 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 possible 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) eburicol a (XI) 4,4,1 Ja-trimethyl-5a-stigmasta-8,Z-24(24')-dien-3~-ol (XII) 4,4-dimethyl-5a-ergosta-8,24(24')-dien-3P-o1 (XIII) 4,4-dimethyl-5a-stigmasta-8,2-24(24')-dien-3P-o1 (XIV)

4a-methyl sterols 4-methyl-5a-stigmasta-8,Z-24(24')-dien-3~-oI (XV) xxx

4-desmethyl sterols zymosterol" (IX) 5n-cholesta-7,24-dien-3~-ol (X) 5a-stigmasta-8,Z-24(2J1)-dien-3P-ol (XVIII) A'-avenasterol* (XIX) ergosterol" (XX) 5a-stigrnasta-5,7,E-22-trien-3P-o1 (XXI) fecosterol (XVI) 5a-ergosta-8-en-38-01 (XXII) 5a-ergosta-S,E-22-dien-3,8-01 (XXIV) 5a-ergosta-5,8,E-22-trien-3P-o1 (XXVI) 5a-stigmasta-8-ene-3P-01 (XXIII) 5a-stigmasta-8,E-22-dien-3P-ol (XXV) 5a-stigrnasta-5,8&22-trien-3P-o1 (XXVII)

7.5 12.5

3 2 6

6 0.5

19.5 2

22 11 6 2 -

-

-

-

-

-

-

6 3.5 2.5 1 6.5 5 1 -

-

34 -

-

-

2 23 4.5 2 4.5 3 0.5

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 (XI) : zymosterol 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-dienol (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 transformation 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 presenting 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 addition 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 second 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)

x n 1

ASMT

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

H XVI

ASMT

A.SMT -

ASMT --

ASMT

XI1

XIV

xv

XVIII

I i A8-A7-sterol isomerase

,%&

I i A8-A7-sterol isomerase i

'% '

A.SMT '1 A.SMT - - XVII X M

' 1 1 -/ C-5 desaturase #& 'I!: C1-22 desaturase

A24(24l) reductase

HO &-

xx Ho XXI H \

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 observed, 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 incubated with [n~ethyl-~HIAdoMet in the absence of exogenous sterol substrate. After extraction and purification by TLC, sterols were found to be significantly labelled, indicating that microsomes 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

,%,.,&

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

XVI XVIII

.,.,rJs A.SMT - -

HO XVI XVIII

I ~ A24(24')reductase i

LI) LI)

XXII xxm

XXIV XXV

i C-5-desaturase i 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 radioactivity 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 cycloartenol (I) or 24-methylene lophenol (IV), the respective substrates 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-methylene 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. thaliana 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-isomerase from erg6 is present and active in the acetone powder of microsomes from e rg6 4118-pYeDP60. The presence of A8-d7-stero1 isomerase 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 conditions (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 sequences 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 consensus 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 methyltransferase 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 2 4 -Me th y 1 en e lophenol lophenol

24-Methylene

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 determinations. 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 determinations, with IV as reference. Standard deviations were 21 % for V,,,,, (expressed as% V,,,,, ,") and 25% for K,,,.

For VI, 111 and IX, V,,,,, and K,m values 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 polypeptides (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 sequences 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 substrate 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% identical 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 expressed 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) 527

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 described 4-desmethylsterols IX, X, XVIII to XXI [13j, the intermediate 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, respectively. 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 e r o 1 isomerase, was similarly transformed by 4118-pYeDP60 and its sterol composition (Table 1, Fig. 4) totally confirmed the above conclusion. 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 methyltransferase, (SMT, in Fig. l) , a 24-methylene lophenol methyltransferase (SMT,) or a single SMT able to perform both reactions ? When cycloartenol (I) and 24-methylene lophenol (IV) were incubated with AdoMet and the acetone powder of microsomes 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 enzyme encoded by cDNA 411 was 17-times higher for 24-methylene 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 Vmaxs and K,s. This discrepancy between the results obtained with plant enzymatic preparations, which contain either two different SMT or an aspecific one, and the enzymatic product 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.

Considering the kinetic parameters determined with the different plant or yeast sterols for methylation by the A. thaliana SMT encoded by cDNA 411 (Table 2), it is clear that the catalytic 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 substrate, 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 microsomes 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 methylation 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-methylene lophenol C24'-methyItransferase. In contrast we suggest that the first group of cDNAs (R. communis, G. max) could encode a protein involved in the first methyl transfer (C24 methylation), 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 polypeptide encoded by ERG6 is involved in the conversion of zymosterol (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 allowing to show the existence of a specificity for C24 methylation rather than for C24l methylation, and lanosterol is not a physiological substrate in higher plants, which always contain cycloartenol (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 membranes 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 allowing 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 manuscript. 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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Identification of Cdnas Encoding Sterol Methyl-Transferases involved in the Second Methylation Step of Plant Sterol Biosynthesis