BOUVIER-NAVE Et Al. Eur. J. Biochem. 246, 518-529 (1997). Sobrte MS de Esteroles

8/8/2019 BOUVIER-NAVE Et Al. Eur. J. Biochem. 246, 518-529 (1997). Sobrte MS de Esteroles

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

Identification of cDNAs encoding sterol methyl-transferasesinvolved in the second methylation step of plant sterol biosynthesisPierrette BO UVIER-NAV E, Tania HUSSELST EIN , Thierry DESPRE Z2 and Pierre BENVENISTE Institut de B iologie MolCculaire des P lantes , DCpartement dEn zym olog ie Cellulaire et MolCculaire, Institut de Botanique, Strasbourg, FranceLaboratoire 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 forthe formation of 24-alkyl sterols (mainly 24-ethyl sterols) which play major roles in plant growth anddevelopment. The f irst methyl transfer applies to cycloartenol, the second o ne to 24-methylene lophenol.F ive cDNA c lones encoding two Arabidopsis thalianu, tw o Nicotiana tabacum and one Ricinus communisS-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SM T) w ere isolated. Th e deduced aminoacid sequences of A. thaliana and N . tabacum S M T a r e a bout 80 % identical in all possible combinations.In contrast they are about 40% identical with the deduced amino acid sequence of R. communis S M Tand the published Glycine max sequence. Both A. thaliana a nd one N. tabacum S M T c DNAs we r eexpressed in a yeast null m utant erg6, deficient in AdoMet zymosterol C24-methyltransferase and contain-ing C24-non-alkylated sterols. In all cases, several 24-ethylidene sterols were synthesized. A thoroughstudy of the sterolic composition of erg6 expressing the A. thaliana cDNA 411 (erg6-4118-pYeDP60)showed 24-m ethylene an d 24-ethyliden e derivatives of 4-desmethy1, 4a-methyl a nd 4,4-dimethyl sterolsas 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 dem onstrat ed by 40 0 M Hz H NM R .Microsomes from erg-6-42 18-pYeDP6 0 were shown to possess AdoM et-dependent sterol-C-methyl-transferase activity. Delipidated preparations of these microsomes converted cycloartenol into 24-methy-lene cycloartanol and 24-methylene lopheno l into 24-ethylidene lophenol, thus allowing the f irst identifi-cation of a plant sterol-C-methyltransferase cDNA. The catalytic eff iciency of the expressed SMT was17-times higher with 24-methylene lophenol than with cycloartenol. This result provides evidence thatth e A. thuliana c D N A 4 1 1 (and most probably the 3 plant SM T cDN As presenting 80% identity with it)encodes a 24-methylene lophenol-C-24l methyltransferase catalyzing the second methylation step of plantsterol biosynthesis.Keywords: plant sterol methyltransferase; yeast transformation; com plementation analysis; 24-methylenelophenol ; cycloartenol.

Sterols from fungi and higher plants differ from vertebratesterols 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. Thisalkylation of the side chain is catalyzed by S-adenosyl-L-m ethio-nine (AdoMet) sterol C-methyltransferases (SMT). In Saccharo-myces cerevihiue, the S M T converts zymoste rol (IX) into feco-sterol (XVI) [3] (Fig. 3). In higher plants, the presence of 24-ethyl sterols results from tw o distinct methyl transfers fro m Ad-oMet [ l , 21. According to the chemical structures of intermedi-Correspondence to P. Bouvier-NavC, Institut de B iolog ie Moltcula iredes Plantes, DCpartement dEnzymolagie Cellulaire et MolCculaire,Instituf de Botan ique, 28 ru e Goethe, F-67083, Strasbourg CCdex, FranceFax: +33 03 88 35 84 84.U R L : http :llibmp.u-strasb~.~rlAbbreviations. SMT, sterol C-methyltransferase AdoM et, 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 submittedto the GenBanEMBL data bank and are available under accessionnumbers: cDNA 411, X89867; cDNA 205, U71400; cDNA 132,U71108; cDNA 412, U71107; cDNA rmt, U81313.

ates of plant sterol biosynthesis an d substrate-specificity studies,it is generally assumed that cycloartenol (I) (Fig. 1) is the sub-strate of the first methylation reaction , resulting in 24-meth ylenecycloartanol (11) [4-71, where as 24-methylene lophenol (IV) isthe preferred substrate for the second methylation, yielding 24-ethylidene lophenol (V ) [8, 91 (Fig. 1 ) . Because the chemicalstructures of I an d IV are very different, it has been suggestedthat the two methylation reactions would be catalyzed by twodifferent enzymes [9]. However, since no plant SMT has beenpurified so far, the hypothesis of a unique plant S M T catalyzingboth alkylations [lo] should be considered. In a ny case the sec-ond methylation is a unique process, absent in vertebrates andmost fungi, leading to the higher plant 24-ethyl sterols. Thesetypical phytosterols were shown to develop specific interactionswith plant phospholipids [ l l] .Two plant S M T genes were recently cloned and their geneproducts preliminarily characterized [12, 131. Th e f irst reportedplant SM T cDNA was iso la ted f rom Glycine rnax [12, 1 41; thededuced amino acid sequence showed three conserved regionsfound in AdoMet-dependent methyltransferases and 47 % iden-tity with the predicted am ino acid sequence of ERC6, the yeastSMT-encoding gene. The G. max cDNA was expressed inEscherichia coli and shown to possess SM T activity: in the pres-


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Bouvier-NavC et al. ( E m J. Biochem. 246) 519

I n I11

IV

11

V

1111

24-meth yl sterols 24-ethyl sterolsFig. 1. Simplified sterol biosynthe sis pathway in higher plan ts show ing the tw o methylation steps. AdoHCy, S-adenosylhomocysteine;I, cycloartenol = 4,4,14a-trimethyl-9~,19-cyclo-5a-cholest-24-en-3~-o1;1, 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;V, 24-methylene lophenol = 4a-methyl-5a-ergosta-7,24(24')-dien-3P-ol; , 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. coliconverted 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 butshowed only 38% identity with ERG6 This cDNA, termedcDNA 411, was used to transform a wild type S. cerevisiae aswell as the yeast null mutant erg6, which is deficient in the yeastSMT zymosterol C24-methyltransferase ; in both cases, several24-ethyl and 24-ethylidene sterols were synthetized, indicatingthat the cDNA 411 encodes a plant SMT able to perform twosequential methylations at C24 and C24' of the yeast sterols[13]. At this stage we could not identify exactly the enzymeencoded by ORF 4118. It could be (a) a cycloartenol-C24-meth-yltransferase (SMT, in Fig. 1) of low substrate specificity, (b) a24-methylene-lophenol-C241-methyltransferaseSMT, in Fig. 1)of low substrate specificity, or (c) a single SMT able to performboth methylation reactions.

We now report the characterization of the enzymatic productof 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 fromA. thaliana, Nicotiana tabacum and Ricinus communis and thecomparison of their deduced amino acid sequences with thoseof SMT cDNAs from Glycine max 1121 and A. thaliana [I31showed that they are distributed into two distinct groups, oneincluding the R. communis and G. max cDNAs and the other,the A. thaliana and N . tabacum cDNAs. Since all the resultspresented here clearly show that the second group most probablyencodes a 24-methylene lophenol C24'-methyltransferase, thefirst group is suggested to encode a cycloartenol C24-methyl-transferase.

EXPERIMENTAL PROCEDURESStrains, media and culture conditions. Escherichia coli.

XLlblue recA- [recAJ, lac-, endAI, gyrA96, thi, hsdRI7,SupE44, relAl, (F'proAB, lac lq, lacZAMJ5, T nl O )] .Saccharo-myces cerevisiae. erg6 (a) ade5 h is7-2 leu2-3,112 ura3-52 ERG6d : : L E U 2 ; e rg 2 (a) ade5 his7-2 leu2-3,112 ura3-52 ERG2-4 ::LEU2.

Strains transformed with pYeDP6O were grown for 3 days at30C on minimum medium YNB [6.7 g/l yeast nitrogen base,(Difco)] containing suitable supplements (50 pg/ml each). Theculture was centrifuged, the pellet resuspended in a completemedium [ lo g/l yeast extract (Difco), 10g/l bactopeptone(Difco), 20 g/l galactose] and grown overnight at 30C.

Plasmids. The plasmid pYeDP6O [15] was used to transformyeast 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 utilizesan expression cassette including a galactose-inducible hybridpromoter and a phosphoglycerate kinase (PGK) terminator.Gene expression is driven by the upstream activating sequenceof the yeast GAL10 and CYC4 genes.

cDNA libraries. The clone VBVEC07 EMBL: emb]Z342031ATTS 3237 was isolated by the systematic screening ofa 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 inPCR experiments to obtain cDNA 205.

cDNA 411 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


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520 Bouvier-Navt et al. (Eul: J. Biochern. 246)protoplasts. The library was prepared by one of us with LambdaZap I1 (Stratagene). cDNAs were cloned unidirectionally inEcoR1, XIzoI.cDNA rmt was isolated from a library of endosperm andembryo of immature castor fruits (R . communis strain Baker296). This library was made in Lambda Zap I1 by van de Looet al. [18] and cDNAs were cloned in EcoRI, XhoI.Reformatting and cloning SM T 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 PCRamplification using specific primers.

A. thuliana cDNA 41 1 . Specific primers were designed tointroduce a BamHT restriction site immediately upstream of theinitiation codon and a XbaI site immediately downstream of thestop codon. Direct primer: 5'-cggcggatccATG GAC TCT TTAACA CTC TTC-3'. Reverse primer: 5'kggctctagaTCA AGAACT CTC CTC CGG TGA-3'. The SMT was amplified using 25thermal cycles (1 min 93", 2 min 56", 3 min 72") with Thermusaquaticus ( T i q ) DNA polymerase under the standard conditions.The PCR product was subsequently cloned into Bluescript togive pSK 4117. pSK 4117 was linearized with XbaI , bluntedusing Klenow fragment of DNA polymerase I and subsequentlydigested with Bam H I; the resulting DNA insert (41 18) was li-gated into pYeDP60 containing a BamHI site at one end and ablunted EcoRI site at the other. The resulting plasmid was called4118-pYeDP60.

A. thaliuna cDNA 205. Specific primers were designed tointroduce a BumHI restriction site immediately upstream of theinitiation codon and a KpnI site immediately downstream ofthe stop codon. Direct primer: 5'-gccgggatccATG GAC TCGGTG GCT CTC TAC TGC ACC GC-3'. Reverse primer: 5'-gccgggtaccTCA TTC AGA AGC TTT CTC TGG-3'. The SMTcDNA was amplified using 25 thermal cycles (1 min 93", 2 min56", 3 min 72") with Pyrocaccusfuriusus ( P f u ) DNA polymer-ase under the recommended conditions. The PCR product wasdigested with BamHI and KpnI and inserted between the BamHIand KpnI sites of pSK resulting in 2051-pSK. The BamHI, KpnIinsert in pSK was extracted and subcloned into BamHI, KpnI ofpYeDP60 leading to 2051-pYeDP60.N . tabucum cDNA 132. Specific primers were designed tointroduce an EcoRV restriction site immediately upstream of theinitiation codon and a KpnI site downstream of the stop codon.Direct primer: 5'-gccggatatcATG GAC TCT CTC ACTCTC-3'. Reverse primer: 5'-gccgggtaccTTA CTC TTC AGGTTT TCT GCA-3'. The SMT cDNA was amplified using 25thermal cycles (1 min 93", 2 min 56", 3 min 72") with Pfu DNApolymerase in the recommended conditions. pYeDP60 was lin-earized with BamHI, blunted using Klenow fragment of DNApolymerase I and subsequently digested with KpnI . The PCRproduct was inserted between the blunted BarnHI and the KpnIsites 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, rmtand the PCR derivatives 2051 and 1323 was performed with anautomatic sequencer Perkin Elmer model 373 using T3 and T7primers, specific oligonucleotide sequences belonging to the se-quenced gene and a modified Taq polymerase capable of incor-porating fluorescent dNTP. Complete sequencing of both strandsof DNA was performed. All cDNAs were in pSK except 1323which was in pYeDP60.Transformation of yeast. Transformation was performedaccording to Schiestl and Gietz [19] with some modifications.A fresh yeast culture (initial absorbance = 0.2) was grown incomplete medium YPG [10g/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 Mlithium acetate (LiAc) solution in TrisEDTA buffer (1 mMEDTA, 10 mM Tris/HCI, pH 7.5 for transformation of erg6 andpH 5.0 for erg2) and finally resuspended in 200 pl of the samesolution. The strain erg2 had to be sonicated 4 min before trans-formation. Salmon sperm was added as DNA carrier (100 pgfrom a 10 mg/ml solution in TrisEDTA) after sonication (10 s)and boiling (20 min) to the plasmid DNA (1 pg). Competentyeast cells (SO-80 pl) and 50 ml of a 40% poly(ethylene gly-col), 0.1 M LiAc solution in TrislEDTA (p H 7.5 for erg6 andpH 5.0 for erg2) were added. The mixture was incubated 30 minat 30"C, then 15min at 42C. After centrifugation, erg6 cellswere 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 heatshock.Sterol analysis. Sterol isolation from lyophilized yeast cells[20], eparation of 4,4-dimethyl-, 4rx-methyl and 4-desmethylsterols by TLC and their acetylation [21] were performed asdescribed previously. TLC on silicagel plates impregnated withAgNO, 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 threemigrations, 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 sameR , as the the acetate of zymosterol (IX). Steryl acetates wereidentified by GC on a DB-1 capillary column (according to theirrelative retention time to the internal standard cholesterol), thenby 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) equippedwith an on column injector and a capillary column(30 mX0.25 mm internal diameter) coated with DB5 (J & WScientific). Different fragments obtained are designed by theratio 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~-ylcetate(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 cetate (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/?-ylacetate (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 XVwere in good agreement with literature data ([22, 23, 211, re-spectively). structures of XI1 and XIV were deduced from theirfragmentation pattern.4a-Methyl steryl acetate X X X : 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-


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Bouvier-NavC et al. ( E m J. Biochern. 246) 5218,24(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-ylcetate (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-ylcetate (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-ylcetate (XXVII) : 392 (M'-60) (49),377(68), 351(5), 253(54), 211(31), 157(100). Sterols XXII toXXVI 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 chemicalshifts of signals are given in ppm with tetramethylsilane as theinternal standard, J in Hz.Substrates for the enzymatic studies. Potential substrateswere 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 LAB1-4 grown on LAB 170250F [25] (purity, 92%). Zymosterolwas 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 similarlypurified (99%).Subcellular fractionation. erg6-4118-pYeDP60 cells weredisrupted as described [29] except that KCI was omitted in thewashing buffer, and BSA (1%) added in the disruption medium.The homogenate was centrifuged for 10min at 12OOOXg andthe supernatant for 60 min at 1OOOOOXg. The microsomal pelletwas resuspended (5- 0 mg proteidml) in 50 mM Tris/HClpH 7.5 containing 20% (by vol.) glycerol and kept at -80Cfor months without significant loss of activity. Acetone powderof microsomes was prepared as described [30] then resuspendedand kept frozen as microsome preparations.Enzymatic assays. A radiochemical assay was performedwith [methyl-'H]AdoMet according to Fonteneau et al. [8] butat a smaller scale: the incubation mixture (100 pl) contained thesterolic 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 blanksfor microsomal assays, microsomal proteins were omitted. In theblanks for the kinetic studies, acetone powder was present andthe sterolic substrate was omitted. Incubations were carried outat 30C for 4-12 min and stopped by 100 p1 12% ethanolicKOH. Appropriate sterol carriers were then added. The neutrallipids were extracted with hexane and the sterols were purifiedby TLC as described previously [21]. The 4,4-dimethyl-, 4a-methyl and 4-desmethyl sterols were separately scraped off theplate and their radioactivity determined in a liquid-scintillationspectrometer.

A large-scale, high-yield assay was set up fo r GC and GC-MS. When a high quantity of the product(s) of the enzymaticreaction was needed, the incubation mixture (2 ml) had the samecomposition 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) . Sterolswere extracted and purified as in the radiochemical assay, thenacetylated and identified by GC-MS as in the sterol analysissection. 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 infull agreement with literature data ([22, 24, 311, respectively).Fecosteryl acetate (XVI), stigmasta-8,2-24(24')-dien-3D-y1 ce-tate (XVIII) and d7-avenasteryl acetate (XIX) had M S in fullagreement with those of Husselstein et al. [13].

RESULTSIsolation and sequence analysis of SMT cDNAsfrom A. thaliana, N . tabacum an d R. communis

A. thaliana cDNAs. The systematic screening of an A. thali-an u seedlings cDNA library (expressed sequence tag project)resulted in the identification of a cDNA (VBVEC07) havingsignificant 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 cDNAwas truncated at the 5' end. A PCR fragment containing the 5'end of the cDNA was amplified from a DNA sample of thecDNA library using one oligonucleotide primer in antisenseorientation (PB23, 5'-GAGAAGATTCCAGTCTC-3') deducedfrom the sequence of VBVEC07 and an oligonucleotide com-plementary to T3 promoter. The cloned fragment was sequencedand allowed us to reconstruct a full-length cDNA sequence of1249 bp (cDNA 205) and to deduce an ORF encoding a proteinof 359 amino acids.A probe (782 bp) was synthesized from cDNA 205 by PCRusing two oligonucleotide primers deduced from the sequence(PB 22, 5'-ATCTACGAGTGGGGATGG-3'. and PB 23). ThisPCR product was then used to screen a cDNA library of A.thaliana siliques (400000 recombinants) resulting in the isola-tion of a full-length cDNA (411) of 1411 bp encoding a proteinof 361 amino acids, 38% identical with ERG6 and 82% identicalwith 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 domainsof the sequence of cDNA 205. This cDNA probe was used toscreen 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 atruncated cDNA of 1276 bp encoding a protein of 352 aminoacids, 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) thathas significant identity with ERG6 as a template and two oligo-nucleotide primers (PB 70, S-GACTTCATGAAAATGCCATT-3' ; PB 71, S'-GAAGAACATTGGTGTGAAAATCTC-3') de-duced from putative conserved domains of T 23248. This cDNAprobe was used to screen 500000 recombinants from a castor


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52 2 Bouvier-Navt et al. (EUK . Biochem. 246)2132-1 12412-5 12205-1 12411-2 1Zrmt-5 1Zsmt-2 1Z e r g 6 12132-1 532412-5 472411-2 522205-1 52Zrmt-5 3 4Zsmt-2 55Z e r g 6 4 82132-1 1152412-5 11 02205-1 1152411-2 115Zrmt-5 92Zsmt-2 1 13Zerg6 1112132-1 1782412-5 1732205-1 1782411-2 177Zrmt-5 155Zsmt-2 176Zerg6 1742132-1 2412205-1 2 412412-5 2362411-2 240Zrmt-5 218Zsmt-2 239Zerg6 237

- Q K P E K Y H

A V D L I G V K P G A R IA 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 VA 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 VA 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 VL 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 FI T R G K E L R N I A G V D K T C N FE L Y R P E D PE L Y N S D D PE K Y R D D D EE K F K A E D DD S F D P N N QD S F D P Q N PD K P D E N N P

V E I I H GV 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 M412-5 286 - - - - - R L K M G R I A P W R N H I V V T V L S W L H M205-1 291 - - - - - R L K M G R I A P W R N H V V V V I L S A I n u411-2 290 - - - - - R L K M G R L A Y W R N H I V V Q I L S A V H Mrmt-5 274 F Fsmt-2 295 P Ferg6 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 LQ P S L T G - F R L A I G R F T R N M I K A L F AH F S L S S - F R L T A V O L T K N M V K L E Y V2132-1 3 4 9 I L C R B E - - - - - - - - - - - - -2412-5 3 4 4 I L C R E E H - - - - - - - - - - - -2205-1 3 4 9 I L C R E K A S E - - - - - - - - - -2411-2 3 4 8 I L C R E S P E E S S - - - - - - - -Zrmt-5 3 3 6 F L A Q H S E N Q - - - - - - - - - -Zsmt-2 35 7 F L A R D L D R N - - - - - - - - - -Z e r g 6 3 6 3 F V A R E N A E T P S Q T S Q E A T QFig.2. Sequence alignment of the sterol methyltransferases. Alignment was performed using the PILEUP program of the UCCG packagerun with the default parameters Positions with a consensus residue present in the seven sequences are boxed. 2132-1 and 2412-5 stand for twoSM T found in N tubacum (this work) 2205-2 an d 2411-2 stand for two SM T found in A. thalianu (this work) Zrmt-5, R. communis SMT (thiswork). Zsmt-2, G. mux SM T [12]. Zerg6, S. cerevzszue SM T [ 3 3 ] .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 toa full-length cDNA of 1328 bp encoding a protein of 346 aminoacids, 39% identical with 411, 51% identical with the proteinencoded by ERG6.

Comparison of deduced aminoacid sequences. The fiveabove-mentioned amino acid sequences were aligned using thePile-up program and compared with the amino acid sequencededuced from the G. m a cDNA encoding an AdoMet: AZ4-ste-rol-C-methyltransferase [121 and the amino acid sequence ofERG6 (Fig. 2).

This comparison reveals two main features. These sequencespresent highly homologous regions : one of them (IN)LD(A/V)-GCG(V/I)GGP corresponds to the consensus motif described byseveral authors [35-371 for all AdoMet-dependent 0-,N- andC-methyltransferases catalyzing methyl transfer on e.g. caffeicacid 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 actingon a sterol substrate. Moreover, these seven amino acid se-quences can be divided in at least two groups: the first onecontains the G. max [I21 and the R. communis methyl-transferases, the second one contains the four methyltransferasesfrom A . thaliana and N . tabacum (205, 411, 132, 412). SMTfrom the second group are more than 80% identical in all pos-sible combinations but are less than 40% identical with SMTfrom the first group. In this first group R. communis SMT is83 % identical with G.max SMT. Whereas SMT from the secondgroup possess a hydrophobic domain of approximately 25 aminoacids at the N-terminal position, G. max and R. communis SMTare devoid of such a hydrophobic domain. The yeast SMTencoded by ERG6 is 50% and 38 % identical with plant SMT ofthe first and second group, respectively. In addition, ERG6 SMThas no hydrophobic domain at the N-terminal position. There-fore ERG6 is closer to the first group than to the second.


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Bouvier-NavC et al. (Eul: J. Biochenz. 246) 523Table 1. Sterol composition of mutant yeast strains erg6 and erg2 transformed with plasmid pYeDP60 with or without ORF 4118. Resultsare given as percentages of the total sterol content. Sterols were identified by their RRT in GC an d fragmentation pattern in GC-MS.Sterol class Sterol compou nd Comp osition of

ergb- erg6-4118- erg2- erg2-4118-pYeDP60 pYeDP60 pYeDP60 pYeDP60% of total

4,4-dimethyl sterols lanostero l" (VI)eburicol a (XI)4,4,1Ja-trimethyl-5a-stigmasta-8,Z-24(24')-dien-3~-olXII)4,4-dimethyl-5a-ergosta-8,24(24')-dien-3P-o1XIII)4,4-dimethyl-5a-stigmasta-8,2-24(24')-dien-3P-o1XIV)4a-methyl sterols 4-methyl-5a-stigmasta-8,Z-24(24')-dien-3~-oIXV)xxx4-desmethyl sterols zymo sterol" (IX)5n-cholesta-7,24-dien-3~-olX)5a-stigmasta-8,Z-24(2J1)-dien-3P-olXVIII)A'-avenasterol* (XIX)ergosterol" (XX)5a-stigrnasta-5,7,E-22-trien-3P-o1XXI)

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-o1XXVI)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)

7.512.532660.5

19.52221162-------

63.52.516.551--

34---2234.524.530.5

Lanosterol = 4,4,14a-trirnethyl-5n-cholesta-8,24-dien-3,!-01VI); eburicol = 4,4,14a-trimethyl-5a-ergosta-8,24(24')-dien-3P-olXI ) : zymo-sterol 1 a-cholesta-8,24-dien-3P-o1IX); 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-o1XX).

Expression ofA. thulianu SMT cDNA 411 in erg6. The steroliccomposition of erg6-4118-pYeDP60, first described in Hus-selstein et al. [13], was determined at higher scale in order todetect minor compounds and to further characterize the majorcompounds. In addition to the 4-desmethyl sterols IX, X, XVIII,XIX, XX and XXI [I31 several precursors were identified byGC and GC-MS (Table 1 and Fig. 3).

Sterols XI and XI1 are the 24-methylene and 24-ethylidenederivatives, respectively, of lanosterol (VI). Sterols XI11 andXIV 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-MSof which corresponds to the skeleton of XV with an additional-CH,- in the side chain. The M S does not allow us, however, tolocalize in the side chain the extra methylene; sterol XXX mightbear, on C24, an isopropylidene, isopropenyl or propenyl group.

Although stigmasta-8,24(24')-dienol (XVIII) is the majorsterol of erg6-4118-pYeDP60, it could not be easily isolatedfrom this strain for NMR analysis because of the presence of itsA7-isomer (XIX) and zymosterol (IX) which migrated withXVIII 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 hasbeen postulated that the two methylation reactions occurring inhigher 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. tabacumsuggested they might encode each of these two enzymes. Tocheck this hypothesis these cDNAs were expressed in the null

mutant erg6. To be able to compare reliably results of expressionexperiments, cDNAs 411, 205 and 132 were formatted iden-tically and inserted in pYeDP60 (see Experimental Procedures)resulting in erg6-2051-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 (datanot 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 totalsterols. These results strongly support the idea that A. thalianacDNAs 411 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 lacksAx--d7-~tero1somerase [40]. The sterol composition of erg2-4118-pYeDP60 is shown in Table 1, together with that of erg2transformed with the void plasmid. Sterol XVIII was also themajor sterol in erg2-4118-pYeDP60 and neither sterol XIX norzymosterol (IX) were detected, thus allowing the purification ofXVIII 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, H l g ) , 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 ofSchmitt and Benveniste [21].The sterol composition of erg2 transformed with the voidplasmid is similar to that described for the first isolated erg2strain [41]. It contains mostly 4-desmethyl sterols: XVI, XXII,


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524 Bouvier-NavC et al. (Eul: J . Biochem. 246)

x n 1

ASMT

...,,&A.SMT- L / )H XV I

ASMT

A.SMT-ASMT-

ASMT

XI1

XI V

xv

XVIIIIi8-A7-sterol isomerase,%&A8-A7-sterol somerasei '%

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

' 1 1-/C-5 desaturase# &I!: C1-22desaturaseA24(24l) reductaseHO& -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 werefound in erg6-4118-pYeDP60(Table 1)with th e exception of sterols XVI an d 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 fractionof erg2-4118-pYeDP60, in addition to these four sterols of theergosta-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 whenthey were transformed with the A. thaliana SMT. Thus, in twodifferent yeast strains transformed with ORF-4118, it was shownthat A . thulium SMT (a) can accept four different tetracyclicskeletons, 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 bothtransformed strains further suggests that the A. thaliana enzyme

could catalyze, in certain circumstances, a third methylation ofthe side chain.Characterization and substrate specificity of A. thalianaSM T from erg6-4118-pYeDP60. In preliminary experiments,microsomal preparations from erg6-4118-pYeDP60 were incu-bated with [n~ethyl-~HIAdoMetn the absence of exogenous ste-rol substrate. After extraction and purification by TLC, sterolswere found to be significantly labelled, indicating that micro-somes from erg6-4118-pYeDP60 do contain a SMT activity andthat the enzyme uses as substrates the endogenous sterols associ-ated with the microsomal membranes.

In contrast, microsomal preparations from erg6 transformedwith the void plasmid (erg6-pYeDP60) were devoid of SMT ac-


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Bouvier-NavC et al. (Eur J . Biochem. 246) 525

,%,.,&erg2...........MT ,& A.SMT- m - -.SMTXVI XVIII

.,.,rJsA.SMT- -HO XVI XVIII

IA24(24')reductaseiLI) LI)

XXII xxm

XXIV XXVi C-5-desaturasei I&&

HO HOXXVI 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 biosynthesispathways upstream from zymosterol are identical to those in erg6 and erg6-4118-pYeDP60, respectively (Fig. 3) .

tivity. Indeed no radioactivity was incorporated in the sterolsafter incubation with labelled AdoMet, in agreement with theabsence 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 themethylation of exogenous sterol substrates. To eliminate theseendogenous sterols, the microsomes of erg6-4118-pYeDP60were delipidated with cold acetone. When the resulting acetonepowder was incubated with labelled AdoMet, no significant ra-dioactivity was incorporated in sterols if no exogenous sterolswere added; the specific activity of SMT in acetone powderpreparations, measured with 24-methylene lophenol, was as highas that of microsomes. The acetone powder was thus used in allfurther studies.

Conditions under which A. thaliana SMT activity of acetonepowder from erg6-4118-pYeDP60 was proportional to time andprotein 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 substrateswere then determined using the radiochemical assay (Table 2).Cycloartenol (I ) and 24-methylene lophenol (IV), were firstcompared. While the K,, for IV (5 pM ) was half that for I, theV,,, 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, indicatedby the ratio V,,,IK,,, is about 17-times higher with 24-methylenelophenol (IV) than with cycloartenol (I).

Lanosterol (VI) and obtusifoliol (111) were then comparedwith 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,, bu t a V,,, valueabout half that of I.

The sterol composition of erg6-4118-pYeDP60 (Table 1)clearly suggested that zymosterol (IX), the major sterol of themutant erg6, is significantly methylated by the A. thaliana SMTsince products XVIII to XXI accumulated. In vitro, zymosterolalso proved to be a good substrate of A. thaliana SMT since itsefficiency of methylation (V,,,,,IK,,,) was 71% that of 24-methy-lene lophenol (Table 2).Identification of the methylation product(s) of 24-methylenelophenol, cycloartenol and zymosterol by the A. thalianaSMT. Under conditions where enough product was formed toallow GC and GC-MS analysis, we could clearly confirm that24-methylene lophenol (IV) was transformed in 24-ethylidenelophenol (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) andd7-avenasterol (XIX). This result, clearly confirms that A. thali-ana SMT can perform two successive methylations, allows usto complete the sterol biosynthesis scheme shown in Fig. 3 since


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526 Bouvier-Navt et al . (Eul: J , Biochem. 246)A II

Fig. 5. Gas chromatograms of sterols I, IV and IX before ( t= 0) orafter 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 AdoM et underthe large-scale, high-yield conditions (see Experimental Procedures).Relative retention times (RRT) of steryl acetates on DB-1 column withcholesterol 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 thebiochemical analysis of erg6-4118-pYeDP60 (Table l), and indi-cates that the endogenous AX-A7-isomerase rom erg6 is presentand active in the acetone powder of microsomes from erg64118-pYeDP60. The presence of A8-d7-stero1 somerase activitywas confirmed by (a ) the partial conversion of zymosterol (IX)to cholest-7,24-dienol (X ) when incubated in the absence ofAdoMet and (b ) the conversion of stigmasta-8,24(24')-dienol(XVIII) to A'-avenasterol (XIX) under similar experimental con-ditions (data not shown).

DISCUSSIONSequence comparisons of polypeptides deduced from SMTcDNAs. In the present study we report the isolation of fivecDNA sequences encoding plant AdoMet-dependent SMT. Thisconclusion is based on the identity existing between these se-quences and ERG6, a gene from yeast encoding a zymosterolC24-methyltransferase [32-341. In addition database searchingrevealed similarities between the five polypeptides encoded bythese cDNAs and several other known methyltransferases. In athorough study Kagan and Clarke [ 3 5 ]have reported three con-sensus motifs present in most methyltransferases and probablyinvolved in the binding of AdoMet. The first motif, remarkably

Table 2. Comparison of the apparent kinetic parameters for variouspotential sterolic substrates of the A. thaliana sterol methyl-transferase expressed in erg 6. The kinetic parameters were determinedafter incubation of 10 0 pM [methyl-'H]AdoM et with (i) IV or IX (2-20 pM ) and 0 .06 mg p roteiniml (acetonic pow der from erg6-4118-pYeD P60 m icrosom esj for 4 min, or (iij I, VI o r I11 (4-40 pM ) and0.1 2 mg protein/ml for 13 min. Und er these conditions, the conversionyield 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 determinedK,,, an d V,,,, values are termed apparent.

% relative to pM % relative to24 -Me hy1 n elophenol lophenol24-Methylene24-Methylene lophenol IV" 100 4.9 10 0Cycloartenol I 14 11.4 6Lanosterol VT 1 3 12.4 5Obtusifoliol 111' 7 11.9 3Zymosterol 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 % forV,,,,, (expressed as% V,,,,, ") and 25% for K,,,.Fo r VI , 111and IX , V,,,,, and K,mvalues are means of two separatedeterminations, with I or IV as reference. Deviations from the meanswere less than 20%.

conserved over nearly 85 methyltransferase genes, correspondsto the LD(A/V)GCG(V/I)GGP domain in the five SMT polypep-tides (Fig. 2). The second and third motifs, corresponding toNSFDGAYS and VLKPGSMYVSY, respectively, in A. thuliumcDNA 411 are less conserved throughout methyltransferasesthan motif I. After alignment of the deduced sequences of thefive SMT cDNAs cloned in this work plus those of G. rnuxcDNA 1121 and yeast ERG6 we observed that the seven se-quences have common features : the AdoMet-binding motifno. I, a typical domain YE(YIF/W)GWGXSFHF and a totallyconserved motif IEATCHAP. It is tempting to speculate thatthese last two invariant domains may be involved in sterol sub-strate binding and(or) enzymatic catalysis. However the sevencDNAs also present important differences allowing us to dividethem into at least two groups. The first group includes the G.mux and R. communis cDNAs which are 83% identical to eachother. The second group corresponds to the two A. thulium (411,205) and N. tabucum (205, 412) cDNAs which are more than80% 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 whichcould correspond to a transmembrane domain involved in theassociation of these SMT with the endoplasmic reticulum 1421.This hydrophobic domain is not present either in the first groupof cDNAs o r in ERG6 which in many aspects is closer to thefirst group.Functional expression of SM T cDNAs in erg6. The A. thuliumcDNAs 411 and 205 and the N . tabucum cDNA 132 were ex-pressed in the yeast null mutant erg6. Being deficient in AdoMetzymosterol C24-methyltransferase, this mutant is devoid of 24-alkylated sterols (Table 1). Its transformation by any of the threecDNAs resulted in a sterol profile where stigrnasta-8,24(24')-dienol (XVIII) and d'-avenasterol (XIX) were the major sterols.


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Bouvier-NavC e t al . (Eul: J. Biochem. 246) 527In other words, all three cDNAs encode an enzyme which canproduce in vivo 24-ethylidene sterols from 24-non-alkylatedsterols.

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 XIand XI1 correspond to the products of the first and the secondmethylation, respectively, of lanosterol (VI). Although we didnot detect sterols VII and VIII in either erg6-pYeDP60 or er g b41 18-pYeDP60, they are known intermediates of yeast sterolbiosynthesis [43, 441 and hence they are the rational precursorsof XI11 and XIV on the one hand, and XV on the other, respec-tively. These results complement ou r previous study [13] andclearly show that the A. thatiana SMT encoded by cDNA 411is able, to recognize four different tetracyclic skeletons, 4,4,14-trimethyl, 4,4-dimethyl, 4a-methyl or 4-desmethyl sterol, and tomethylate either a 424(25) or a 424(24') double bond.

The yeast null mutant erg2, deficient in ~ 8 - ~ 7 - ~ t e r o 1som-erase, was similarly transformed by 4118-pYeDP60 and its sterolcomposition (Table 1, Fig. 4) totally confirmed the above con-clusion. Since erg2-4118-pYeDP60 was devoid of zymosteroland 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 itsidentification as 5a-stigma~ta-8,Z-24(24~)-dien-3P-01.

The in vitro study of A. thaliana SMT using acetone powdersof microsomes from erg6-4118-pYeDP60 was fully consistentwith the data obtained in vivo: lanosterol and zymosterol wereshown to be substrates when [3H]AdoMet was added (Table 2),and large-scale, high-yield incubation of zymosterol allowed theidentification of its methylation products : fecosterol (XVI) andstigmasta-8,Z-24 (24')-dien-3P-ol (XVIII) (accompanied by their4' counterparts XVII and XIX) (Fig. 5 ) .

The aim of the in vitro study was to answer the followingquestion: 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 andtheir methylation products, 24-methylene cycloartanol (11) and24-ethylidene lophenol (V), respectively, were clearly identifiedby GC-MS. The kinetic parameters of the reaction for these twosubstrates were determined using labelled AdoMet (Table 2).The V,,, l IVmunv ratio was 141100. The K,, measured for I wastwice 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 usingmicrosomes from bramble cells [8 , 91 clearly showed that bothsterols are methylated. In addition, when cycloartenol (I ) andlanosterol (VI) were compared as substrates for methylation withcell-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 thanVI. The same comparison between I and VI as substrates for theerg6-4118-pYeDP60 SMT showed that both sterols had equiva-lent Vmaxs nd K,s. This discrepancy between the results ob-tained with plant enzymatic preparations, which contain eithertwo different SMT or an aspecific one, and the enzymatic prod-uct of A. thaliana cDNA 411 strongly suggests that plants donot contain a unique SMT and that the A. thaliana SMT understudy is a 24-methylene lophenol methyltransferase.

Considering the kinetic parameters determined with the dif-ferent plant or yeast sterols for methylation by the A. thalianaSMT encoded by cDNA 411 (Table 2), it is clear that the cata-lytic efficiency of this enzyme depends more on the sterolicskeleton than on the position 424(25) or 424(24') of the doublebond in the side chain. Thus zymosterol (IX) is methylated 70%as efficiently as 24-methylene lophenol although it possess aA24 double bond instead of a A24(24') double bond. Hence thepoor methylation efficiency observed with cycloartenol (I) andlanosterol (VI) would not be due to their A24 double bond butto 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 themethylation 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-pYeDP60shows selectivity towards 24-methylene lophenol in vitro andproduces in vivo the accumulation of 24-ethyl sterols in botherg6-4118-pYeDP60 and erg2-4118-pYeDP60, we can concludethat the cDNA 411 encodes a SMT involved in the second meth-ylation step, that is the conversion of methylene-24 lophenolinto 24-ethylidene lophenol. For reasons developed above, sucha conclusion could be extrapolated to the A. thaliana cDNA 205and the N . tabacum cDNA 132, since these cDNAs are morethan 80 % identical to 411 and null mutant erg6 transformedwith plasmids 2051 -pYeDP60 and 1323-pYeDP60 became ableto 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 suggestthat the first group of cDNAs (R. communis, G. max) could en-code a protein involved in th e first methyl transfer (C24 methyl-ation), that is to say catalyzing the conversion of cycloartenolinto 24-methylene cycloartanol (Fig. 1). This hypothesis rests onthe finding that the first group of cDNAs is more similar toERG6 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 ; moreoverERG6 polypeptide seems unable to achieve a C24' methylation,since two 24-methylene sterols, 24-methylene cholesterol andfecosterol (XIV) were shown not to be substrates [46j. In thiscontext it should be recalled that the G. max cDNA has beenexpressed in E. coli as a fusion protein which was shown tocatalyze the conversion of lanosterol (VI) to eburicol (XI) andtherefore to catalyze a C24 methylation [14]. However, such aresult does not constitute a strong argument in favour of ourhypothesis since there are no comparative measurements allow-ing to show the existence of a specificity for C24 methylationrather 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 benecessary to clarify this point.

We believe that the cloning of a 24-methylene lophenolC24'-methyltransferase catalyzing the second methylation stepduring 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 unravellingthe intricate mechanism which controls the alkylation level inhigher plant sterols. This point is important since plant mem-branes contain a mixture of C,,, C,, and C,, sterols differing bytheir methylation extent at C24 and in a defined proportionwhich is inheritable in a given genotype.

We are grateful to Dr M. Bard (Indiana University-Purdu e Univer-sity Indianapolis) for kindly providing the yeast null mutants erg6 and


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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 (Gen Bank ID, T 23248) and for acDNA library from R. communis. We thank also Dr D. Pompon [CentreNational de la Recherche Scientfique (CNRS ), Gif sur Yvette] for allow-ing us to use plasmid pYeDP60, Dr J . Giraudat (CN RS, Gif-sur-Yvette)for giving us a cDNA library of A. thalianu siliques and P rofs T. J. Bachand M. Rohmer (CNRS, Strasbourg) for carefully reading the manu-script. We warmly acknowledge the skillful assistance of P. Hamann forthe cDNA sequencing, R. Meens for the GC-MS and B. Bastian whopatiently typed the manuscript.

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try 21, 1959-1967.

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