Characterization of cyclopropane fatty-acid synthase from Sterculia foetida
Characterization of cyclopropane fatty-acid synthase from Sterculia foetida
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DOI：
10.1074/jbc.m212464200
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发表时间：
2003-04-11
影响因子：
4.8
通讯作者：
Ohlrogge, JB
中科院分区：
文献类型：
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作者：
Bao, XM;Thelen, JJ;Ohlrogge, JB
发育期种子中的环丙烷合成酶催化来自S-腺苷蛋氨酸的亚甲基加成到油酸的顺式双键上(Bao，X.，Katz，S.，Pollard，M.，and OhlRogge，J.(2002)Proc.Natl ACA&Sci.美国A.99,7172-7177)。为了更好地了解该酶，研究了植物环丙烷合成酶在叶片和种子组织中的差异表达、蛋白质性质和底物选择性。免疫印迹分析表明，重组赤霉环丙烷合成酶(SfCPA-Fas)在种子中表达，但在叶片中不表达，是一种定位于微粒体部分的膜蛋白。在体内用L-[甲基-C-14]蛋氨酸标记转化烟草细胞，用S-腺苷-L-[甲基-C-14]蛋氨酸进行体外检测。这些动力学实验表明，油酸在磷脂酰胆碱(PC)的sn-1位上进行酯化反应，合成了二氢脂肪酸酯。此外，对赤霉菌种子发育中的PC和表达SfCPA-Fas的烟草细胞中积累的sn-1和sn-2位的酰基链的分析也表明，90%以上的二氢胸酯酸酯化到sn-1位。因此，我们认为SfCPA-Fas是一种微粒体定位的膜蛋白，它催化S-腺苷-L-蛋氨酸衍生的亚甲基通过油酸的双键酯化到PC的sn-1位。对植物和细菌基因组中与SfCPA-Fas相关的序列的研究表明，带有黄素结合位点的多肽结构域要么与植物蛋白的甲基转移酶结构域融合，要么通常由细菌环丙烷合成酶基因附近的基因编码。
Cyclopropane synthase from Sterculia foetida developing seeds catalyzes the addition of a methylene group from S-adenosylmethionine to the cis double bond of oleic acid (Bao, X., Katz, S., Pollard, M., and Ohlrogge, J. (2002) Proc. NatL Aca& Sci. U. S. A. 99, 7172-7177). To understand this enzyme better, differential expression in leaf and seed tissues, protein properties, and substrate preferences of plant cyclopropane synthase were investigated. Immunoblot analysis with antibodies raised to recombinant S. foetida cyclopropane synthase (SfCPA-FAS) revealed that SfCPA-FAS is expressed in S. foetida seeds, but not in leaves, and is a membrane protein localized to microsomal fractions. Transformed tobacco cells expressing SfCPA-FAS were labeled in vivo with L-[methyl-C-14]methionine and assayed in vitro with S-adenosyl-L-[methyl-C-14]methionine. These kinetic experiments demonstrated that dihydrosterculate was synthesized from oleic acid esterified at the sn-1 position of phosphatidylcholine (PC). Furthermore, analysis of acyl chains at sn-1 and sn-2 positions that accumulated in PC from S. foetida developing seeds and from tobacco cells expressing SfCPA-FAS also demonstrated that greater than 90% of dihydrosterculate was esterified to the sn-1 position. Thus, we conclude that SfCPA-FAS is a microsomal localized membrane protein that catalyzes the addition of methylene groups derived from S-adenosyl-L-methionine across the double bond of oleic acid esterified to the sn-1 position of PC. A survey of plant and bacterial genomes for sequences related to SfCPA-FAS indicated that a peptide domain with a putative flavin-binding site is either fused to the methyltransferase domain of the plant protein or is often found encoded by a gene adjacent to a bacterial cyclopropane synthase gene.