Plant sterol biosynthesis:: identification of two distinct families of sterol 4α-methyl oxidases
Plant sterol biosynthesis:: identification of two distinct families of sterol 4α-methyl oxidases
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DOI：
10.1042/bj20031572
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发表时间：
2004-03-15
影响因子：
4.1
通讯作者：
Rahier, A
中科院分区：
文献类型：
--
作者：
Darnet, S;Rahier, A
在植物中，将环阿屯醇转化为功能性植物固醇需要通过包括固醇4 α-甲基氧化酶（SMO）的酶复合物去除C-4处的两个甲基。我们报告的候选基因的克隆拟南芥中的SMO，属于两个不同的家庭称为SMO 1和SMO 2，并分别含有三个和两个异构体。SMO 1和SMO 2的序列同源性较低，与酿酒酵母的ERG 25基因同源性较低。植物SMO氨基酸序列具有所有三个富含组氨酸的基序（HX 3 H、HX 2 HH和HX 2 HH），这是参与脂质氧化的膜结合非血红素铁加氧酶小家族的特征。为了阐明SMO I和SMO 2基因家族的确切功能，我们通过使用VIGS（病毒诱导的基因沉默）方法在本氏烟草中降低了它们的表达。将SMO 1和SMO 2的cDNA片段插入病毒载体，N.接种病毒转录物的本萨米亚那。在用SMO 1沉默后，获得了4，4-二甲基-9 β，19-环丙基甾醇（即24-亚甲基环木菠萝烷醇）的大量积累，而4 α-甲基甾醇的定性和定量水平不受影响。在用SMO 2沉默的情况下，发现4 α-甲基-δ（7）-甾醇（即24-亚乙基苯酚和24-乙基苯酚）的大量积累，而4，4-二甲基甾醇的水平没有变化。这些清楚和不同的生化表型表明，与动物和真菌相比，在光合真核生物中，这两个新的cDNA家族编码两种不同类型的C-4-甲基甾醇氧化酶，分别控制4，4-二甲基甾醇和4 α-甲基甾醇前体的水平。
In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO. The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation. To elucidate the precise functions of SMO I and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana. SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N. benthamiana inoculated with the viral transcripts. After silencing with SMO1, a substantial accumulation of 4,4-dimcthyl-9beta,19-cyclopropyl sterols (i.e. 24-methyl-enecycloartanol) was obtained, whereas qualitative and quantitative levels of 4alpha-methylsterols were not affected. In the case of silencing with SMO2, a large accumulation of 4alpha-methyl-Delta(7)-sterols (i.e. 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols. These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4alpha-methylsterol precursors respectively.