Cloning of the gene for monogalactosyldiacylglycerol synthase and its evolutionary origin.
Cloning of the gene for monogalactosyldiacylglycerol synthase and its evolutionary origin.
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DOI：
10.1073/pnas.94.1.333
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发表时间：
1997-01
影响因子：
11.1
通讯作者：
M. Shimojima;Hiroyuki Ohta;Akihiro Iwamatsu;T. Masuda;Y. Shioi;K. Takamiya
中科院分区：
文献类型：
--
作者：
M. Shimojima;Hiroyuki Ohta;Akihiro Iwamatsu;T. Masuda;Y. Shioi;K. Takamiya
单半乳糖二酰甘油(MGDG)合成酶(UDP半乳糖：1，2-二酰甘油3-β-D-半乳糖基转移酶；EC 2.4.1.46)催化叶绿体的主要结构脂--MGDG的形成。我们从黄瓜cdna文库中克隆了该合酶的cdna。该克隆全长2142bp，含有一个1575bp的开放阅读框，编码525aa。开放阅读框由成熟蛋白(422个氨基酸；Mr为46,552)和转运肽(103个氨基酸)组成。虽然成熟蛋白区的分子量与黄瓜子叶纯化的相同，但与其他研究小组报道的菠菜成熟蛋白的分子量(约20 kDa)有很大的不同。该蛋白成熟区在大肠杆菌中以谷胱甘肽S转移酶融合蛋白的形式表达。在大肠杆菌中的表达表明，该蛋白能高效催化MGDG的合成。因此，我们推测该基因编码黄瓜MGDG合成酶。此外，MGDG合成酶的推导氨基酸序列与枯草芽孢杆菌和大肠杆菌的MURG序列具有同源性，MUG编码一种糖基转移酶，催化细菌合成肽聚糖的最后一步。这一序列同源性表明，叶绿体膜生物合成的机制是从细菌的细胞壁生物合成进化而来的。这与叶绿体形成的内共生假说是一致的。
Monogalactosyldiacylglycerol (MGDG) synthase (UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46) catalyzes formation of MGDG, a major structural lipid of chloroplast. We cloned a cDNA for the synthase from cucumber cDNA library. The full-length cDNA clone was 2142 bp, and it contains a 1575-bp open reading frame encoding 525 aa. The open reading frame consists of the regions for a mature protein (422 aa; Mr of 46,552) and transit peptide to chloroplast (103 aa). Although the molecular weight of mature protein region matched that purified from cucumber cotyledons, it was quite different from those purified from spinach (approximately 20 kDa) reported by other groups. The mature region of the protein was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The expression in E. coli showed that the protein catalyzed MGDG synthesis very efficiently. Therefore, we concluded that the cDNA encodes MGDG synthase in cucumber. In addition, the deduced amino acid sequence of the MGDG synthase cDNA showed homology with MurG of Bacillus subtilis and E. coli, which encode a glycosyltransferase catalyzing the last step of peptidoglycan synthesis in bacteria. This sequence homology implies that the machinery of chloroplast membrane biosynthesis is evolutionarily derived from that of cell wall biosynthesis in bacteria. This is consistent with the endosymbiotic hypothesis of chloroplast formation.