Isolation of a gene encoding a 1,2-diacylglycerol-sn-acetyl-CoA acetyltransferase from developing seeds of Euonymus alatus
Isolation of a gene encoding a 1,2-diacylglycerol-sn-acetyl-CoA acetyltransferase from developing seeds of Euonymus alatus
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DOI：
10.1074/jbc.m410276200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Pollard, M
中科院分区：
文献类型：
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作者：
Milcamps, A;Tumaney, AW;Pollard, M
1,2-二酰基-3-乙酰基- asn -甘油（ac-TAG）是一种罕见的三酰基甘油，是火种卫矛种子中主要的储存脂质。这些acTAGs在甘油的sn-1和sn-2位置都有酯化的长链酰基。长链酰基辅酶a和乙酰基辅酶a sn-1,2-二酰基甘油酰基转移酶（DGAT）均具有活性。我们从卫矛的发育种子中分离出一个基因，显示出与DGAT1基因家族成员非常高的序列相似性。与酰基辅酶胆固醇酰基转移酶有关)。当该基因在野生型酵母中表达时，会导致长链三酰甘油（Ic-TAG）积累增加5倍，并出现低水平的ac-TAG。采用气相色谱-质谱联用技术鉴定了氢化ac-TAG的分子种类。从该转化酵母中分离的微粒体显示二酰基甘油：乙酰辅酶a乙酰转移酶活性，比用空载体或用拟南芥DGAT1基因转化的酵母制备的微粒体高约40倍。该微粒体乙酰转移酶活性的具体活性与用空载体或拟南芥或羽onymus DGAT1基因转化的酵母系的微粒体长链DGAT活性的测定具有相同的数量级。尽管如此，酵母中ac-TAG的积累量很低（占Ic-TAG的0.26%），而Ic-TAG的积累量却增加了。讨论了这种异常的可能原因。在删除内源TAG合成的酵母细胞系中表达了卫矛dgat1样基因，证实了该基因产物具有长链和乙酰转移酶活性。
1,2-Diacyl-3-acetyl-sn-glycerols (ac-TAG) are unusual triacylglycerols that constitute the major storage lipid in the seeds of Euonymus alatus (Burning Bush). These acTAGs have long-chain acyl groups esterified at both the sn-1 and sn-2 positions of glycerol. Cell-free extracts of developing seeds of E. alatus contain both long-chain acyl-CoA and acetyl-CoA sn-1,2-diacylglycerol acyltransferase (DGAT) activity. We have isolated a gene from developing seeds of Euonymus alatus that shows a very high sequence similarity to the members of the DGAT1 gene family (ie. related to acyl-CoAcholesterol acyltransferases). This Euonymus DGAT1 gene, when expressed in wild type yeast, results in a 5-fold enhancement of longchain triacylglycerol (Ic-TAG) accumulation, as well as the appearance of low levels of ac-TAG. Hydrogenated ac-TAG molecular species were identified by gas chromatography-mass spectrometry. Microsomes isolated from this transformed yeast show diacylglycerol:acetyl-CoA acetyltransferase activity, which is about 40-fold higher than that measured in microsomes prepared from yeast transformed with the empty vector or with the Arabidopsis thaliana DGAT1 gene. The specific activity of this microsomal acetyltransferase activity is of the same order of magnitude as the microsomal long-chain DGAT activities measured for yeast lines transformed with the empty vector or either the Arabidopsis or Euonymus DGAT1 genes. Despite this, ac-TAG accumulation in yeast transformed with the Euonymus DGATI gene was very low (0.26% of Ic-TAG), whereas Ic-TAG accumulation was enhanced. Possible reasons for this anomaly are discussed. Expression of the Euonymus DGAT1-like gene in yeast lines where endogenous TAG synthesis has been deleted confirmed that the gene product has both long-chain and acetyltransferase activity.