A 24-methylene lophenol C-28 methyltransferase from suspension cultures of bramble cells

Fonteneau, Paul; Hartmann-Bouillon, Marie-Andrée; Benveniste, Pierre

doi:10.1016/0304-4211(77)90122-5

Cited by 47 publications

(23 citation statements)

References 11 publications

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“…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 consistently a better substrate than VI.…”

Section: Discussionmentioning

confidence: 94%

“…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.…”

Section: Strainsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Bouvier-Navé

Husselstein

Desprez

et al. 1997

European Journal of Biochemistry

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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 Saccharomyces 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 AdoMet [l, 21. According to the chemical structures of intermediCorrespondence to P.

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Section: Discussionmentioning

confidence: 94%

Section: Strainsmentioning

confidence: 99%

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

Bouvier-Navé

Husselstein

Desprez

et al. 1997

European Journal of Biochemistry

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“…The presence in higher plants of 24-ethyl sterols corresponds to two distinct methyl transfers from AdoMet [1,2]. In agreement with the chemical structures of intermediates of plant sterol biosynthesis, it was shown by substrate-specificity studies that cycloartenol (I) is the substrate of the first methylation reaction, resulting in 24-methylene cycloartanol (II) [3Ϫ6], whereas 24-methylene lophenol (IV) is the preferred substrate for the second methylation, yielding 24-ethylidene lophenol (V) [7,8] Correspondence to P. …”

mentioning

confidence: 84%

Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis

Bouvier-Navé¹,

Husselstein²,

Benveniste³

1998

European Journal of Biochemistry

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Two methyl transfers are involved in the biosynthesis of 24-methyl and 24-ethyl sterols, which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second to 24-methylene lophenol. About ten cDNA clones encoding S-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SMTs) have been isolated so far from various plants. According to their deduced amino acid sequences, they were classified in two families, smt1 and smt2; in addition, smt2 cDNAs were shown to encode a 24-methylene lophenol C24 1 methyltransferase [Bouvier-Navé, P., Husselstein, T., Desprez, T. & Benveniste, P. (1997) Eur. J. Biochem. 246, 518Ϫ529]. We now report the comparison of two cDNAs isolated from Nicotiana tabacum, Ntsmt1-1 which belongs to the first SMT cDNA family and Ntsmt2-1 which belongs to the second. Both cDNAs were expressed in the yeast null mutant erg6, deficient in SMT. Whereas erg6 is devoid of 24-alkyl sterols, erg6 Ntsmt1-1 contained a majority of 24-methylene sterols and erg6 Ntsmt2-1, a majority of 24-ethylidene sterols, indicating distinct functions for the expression products of these cDNAs. In the presence of AdoMet, delipidated microsomes from erg6 Ntsmt1-1 efficiently converted cycloartenol into 24-methylene cycloartanol, but did not produce any 24-ethylidene lophenol upon incubation with 24-methylene lophenol. This demonstrates that cDNA Ntsmt1-1 (and most probably the other plant SMT cDNAs of the first family) encode(s) a cycloartenol C24 methyltransferase. In contrast, delipidated microsomes of erg6 Ntsmt2-1 were shown to methylate preferentially 24-methylene lophenol, as expected from an SMT encoded by an smt2 cDNA. In summary, among various cDNAs isolated from N. tabacum, one (Ntsmt1-1) belongs to the first family of plant SMT cDNAs according to its deduced amino acid sequence and was shown to encode a cycloartenol C24 methyltransferase, whereas another (Ntsmt2-1) belongs to the second family and was shown to encode a 24-methylene lophenol C24 1 methyltransferase. Meanwhile, two cDNAs were isolated from Oriza sativa and shown to belong to smt1 and to smt2 families, respectively. These data disclose the coexistence, in a given plant species, of two distinct SMTs, each catalyzing one step of methylation in the sterol biosynthesis pathway.Keywords : plant sterol methyltransferase; yeast transformation; complementation analysis ; cycloartenol; 24-methylene lophenol.Sterols from higher plants and fungi differ from vertebrate sterols by the presence of an extra alkyl group at C24 [1,2]. The alkylation of the side chain of sterols is catalyzed by Sadenosyl-L-methionine (AdoMet) sterol C24 methyltransferases (SMTs). The presence in higher plants of 24-ethyl sterols corresponds to two distinct methyl transfers from AdoMet [1,2]. In agreement with the chemical structures of intermediates of plant sterol biosynthesis, it was shown by substrate-specificity studies that cycloartenol (I) is the substrate of the first methylation reaction, resulting in 24-methylene cycloartan...

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“…In higher plants, the presence of 24-ethyl sterols results from two distinct methyl transfers from S-adenosyl-L-methionine [1,2]. According to the chemical structures of intermediates of sterol biosynthesis and substrate specificity studies, it is generally assumed that cycloartenol (V) is the substrate of the first methylation reaction resulting in 24-methylene cycloartanol (VI) [5,6], whereas 24-methylene lophenol (VII) is the preferred substrate for the second methylation resulting in 24-ethylidene lophenol (VIII) [7] (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

Transformation of Saccharomyces cerevisiae with a cDNA encoding a sterol C‐methyltransferase from Arabidopsis thaliana results in the synthesis of 24‐ethyl sterols

et al. 1996

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Using an EST-cDNA probe, a full-length cDNA (411) sequence of 1411 bp was isolated from A. thaliana. This sequence contained features typical of methyltransferases in general and in particular showed 38% identity with ERG6, a S. cerevisiae gene which encodes the zymosterol-C-24-methyltransferase. A yeast vector containing this ORF (4118-pYeDP60) was used to transform a wild type S. cerevisiae which accumulates predominantly ergosterol, a 24-methyl sterol as well as a mutant erg6 null mutant accumulating principally zymosterol, a sterol non-alkylated at C-24. In both cases, several 24-ethyl-and 24-ethylidene sterols were synthetized indicating that the 4118 cDNA encodes a plant sterol C-methyltransferase able to perform two sequential methylations of the sterol side chain.

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A 24-methylene lophenol C-28 methyltransferase from suspension cultures of bramble cells

Cited by 47 publications

References 11 publications

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

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

Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis

Transformation of Saccharomyces cerevisiae with a cDNA encoding a sterol C‐methyltransferase from Arabidopsis thaliana results in the synthesis of 24‐ethyl sterols

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