Cloning and functional characterization of a Phospholipid:Diacylglycerol acyltransferase from Arabidopsis
Cloning and functional characterization of a Phospholipid:Diacylglycerol acyltransferase from Arabidopsis
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DOI：
10.1104/pp.104.044354
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发表时间：
2004-07-01
期刊：
影响因子：
7.4
通讯作者：
Stymne, S
中科院分区：
文献类型：
--
作者：
Stahl, U;Carlsson, AS;Stymne, S
最近描述了涉及磷脂的三酰基甘油生物合成的新途径：二酰基甘油酰基转移酶（PDAT）（Dahlqvist A，斯塔尔U，Lenman M，Banas A，Lee M，Sandager L，伦讷H，Stymne S，[2000] Proc Natl Acad Sci USA 97：6487-6492）。在酵母（酿酒酵母）中鉴定了编码PDAT的LRO 1基因，并显示其与动物卵磷脂：胆固醇酰基转移酶具有同源性。对拟南芥基因组数据库的搜索鉴定了由At 5g 13640基因编码的蛋白质作为与酵母PDAT最接近的同源物（28%氨基酸同一性）。At 5g 13640（AtPDAT基因）的cDNA在拟南芥中位于花椰菜花叶病毒启动子之后被过量表达。来自过表达者的根和叶的微粒体制剂具有与该基因的表达水平相关的PDAT活性，从而证明该基因编码PDAT（AtPDAT）。AtPDAT利用不同的磷脂作为酰基供体，并接受C10至C22范围内的酰基。活性的速率是高度依赖于酰基组合物与最高的活性为酰基含有几个双键，环氧基，或羟基。该酶利用磷脂酰胆碱的两个sn-位置，但对sn-2位置具有3倍的偏好。过表达AtPDAT的拟南芥植株的脂肪酸和脂质组成以及单位鲜重的脂质量与野生型没有显著差异。从AtPDAT基因的T-DNA插入突变体的根的微粒体制剂几乎没有检测到的能力，从磷脂酰基转移到添加的二酰基甘油。然而，这些微粒体仍然能够通过二酰基甘油：二酰基甘油酰基转移酶反应以与野生型微粒体制备物相同的速率进行三酰基甘油合成。
A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487-6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin: cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.