Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration -: The DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development
Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration -: The DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development
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DOI：
10.1074/jbc.m506859200
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发表时间：
2005-10-14
影响因子：
4.8
通讯作者：
Mueller-Roeber, B
中科院分区：
文献类型：
--
作者：
Gómez-Merino, FC;Arana-Ceballos, FA;Mueller-Roeber, B
甘油二酯激酶（DGK）调节第二信使甘油二酯的水平，并产生磷脂酸（PA），另一种信号分子。拟南芥基因组编码7个推定的二酰甘油激酶同工酶（命名为AtDGK 1 - 7），结构上分为三个主要的集群。到目前为止，还没有报道任何植物簇II DGK的酶活性。在这里，我们证明了这个集群的代表，AtDGK 7，是生化活性时，在大肠杆菌中表达的重组蛋白。AtDGK 7基因由At 4g 30340基因座编码，含有374个氨基酸，表观分子量为41.2kDa。AtDGK 7具有N-末端催化结构域，但与各种表征的DGK（包括AtDGK 2）相反，其在其N末端缺乏富含半胱氨酸的结构域，并且重要的是，其C-末端DGK辅助结构域是不完整的。在大肠杆菌中表达了AtDGK 7。大肠杆菌以1，2-二油酰-sn-甘油为底物，表现出米氏动力学。AtDGK 7活性受pH、洗涤剂和DGK抑制剂R59022的影响。我们证明了AtDGK 2和AtDGK 7磷酸化通常在植物中发现的二酰基甘油分子种类，这表明这两种酶转化生理相关的底物。AtDGK 7在整个拟南芥植物中表达，但在花和幼苗中表达最强。AtDGK 2的表达由创伤瞬时诱导。R59022在接近80 μ M时抑制根伸长和侧根形成，并降低植物生长，表明DGK在植物发育中起重要作用。
Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at similar to 80 mu M inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development.