Bouvier-Navé P, Husselstein T, Desprez T, Benveniste P
Institut de Biologie Moléculaire des Plantes, Département d'Enzymologie Cellulaire et Moléculaire, Institut de Botanique, Strasbourg, France.
Eur J Biochem. 1997 Jun 1;246(2):518-29. doi: 10.1111/j.1432-1033.1997.t01-1-00518.x.
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 thaliana, 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-desmethyl, 4alpha-methyl and 4,4-dimethyl sterols as well as 24-methyl and 24-ethyl derivatives of 4-desmethyl sterols. The structure of 5alpha-stigmasta-8, Z-24(24(1))-dien-3beta-ol, the major sterol of transformed yeasts, was demonstrated by 400 MHz 1H NMR. Microsomes from erg6-4118-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. thaliana cDNA 411 (and most probably the 3 plant SMT cDNAs presenting 80% identity with it) encodes a 24-methylene lophenol-C-24(1) methyltransferase catalyzing the second methylation step of plant sterol biosynthesis.
植物甾醇生物合成过程涉及两次甲基转移，负责形成24-烷基甾醇（主要是24-乙基甾醇），这些甾醇在植物生长和发育中起主要作用。第一次甲基转移作用于环阿屯醇，第二次作用于24-亚甲基环菠萝醇。分离出了五个编码两种拟南芥、两种烟草和一种蓖麻的S-腺苷-L-甲硫氨酸（AdoMet）甾醇甲基转移酶（SMT）的cDNA克隆。拟南芥和烟草SMT推导的氨基酸序列在所有可能组合中约80%相同。相比之下，它们与蓖麻SMT推导的氨基酸序列以及已发表的大豆序列约40%相同。拟南芥和一种烟草的SMT cDNA均在酵母缺陷型erg6中表达，该酵母缺乏AdoMet酵母甾醇C24-甲基转移酶且含有C24-非烷基化甾醇。在所有情况下，均合成了几种24-亚乙基甾醇化合物。对表达拟南芥cDNA 411（erg6-4118-pYeDP60）的erg6酵母甾醇组成的深入研究表明，存在4-去甲基甾醇、4α-甲基甾醇和4,4-二甲基甾醇的24-亚甲基和24-亚乙基衍生物，以及4-去甲基甾醇的24-甲基和24-乙基衍生物。通过400 MHz 1H NMR确定了转化酵母的主要甾醇5α-豆甾-8,Z-24(24(1))-二烯-3β-醇的结构。erg6-4118-pYeDP60的微粒体显示具有依赖AdoMet的甾醇-C-甲基转移酶活性。这些微粒体的脱脂制剂将环阿屯醇转化为24-亚甲基环阿屯醇，将24-亚甲基环菠萝醇转化为24-亚乙基环菠萝醇，从而首次鉴定出一种植物甾醇-C-甲基转移酶cDNA。表达的SMT对24-亚甲基环菠萝醇的催化效率比对环阿屯醇高17倍。这一结果证明拟南芥cDNA 411（很可能还有与其80%相同的3种植物SMT cDNA）编码一种24-亚甲基环菠萝醇-C-24(1)甲基转移酶，催化植物甾醇生物合成的第二步甲基化反应。
