Regulation of serine biosynthesis in Arabidopsis -: Crucial role of plastidic 3-phosphoglycerate dehydrogenase in non-photosynthetic tissues
Regulation of serine biosynthesis in Arabidopsis -: Crucial role of plastidic 3-phosphoglycerate dehydrogenase in non-photosynthetic tissues
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DOI：
10.1074/jbc.274.1.397
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发表时间：
1999-01-01
影响因子：
4.8
通讯作者：
Saito, K
中科院分区：
文献类型：
--
作者：
Ho, CL;Noji, M;Saito, K
在植物中，丝氨酸通过几种途径合成。3-磷酸甘油酸脱氢酶（PGDH）是参与丝氨酸生物合成磷酸化途径的第一个酶，负责3-磷酸甘油酸氧化为磷酸羟基丙酮酸。本研究首次从拟南芥中克隆了PGDH基因，并对其进行了序列分析。cDNA和基因组克隆的序列分析表明，PGDH基因由3个外显子组成，编码623个氨基酸（66，453 Da）的多肽。推导的蛋白质，包含三个最保守的区域在NAD依赖的2-羟酸脱氢酶家族，有38-39%的同一性，其动物和细菌的对应物。使用绿色荧光蛋白作为亚细胞定位的报告蛋白，通过粒子枪轰击实验证实了用于易位到质体中的N-末端信号序列的存在。Southern杂交分析和限制性片段长度多态性分析表明，PGDH是一个单拷贝基因，定位于1号染色体的上臂。北方杂交分析表明PGDH mRNA在光生植物的根组织中优先表达，表明丝氨酸生物合成的磷酸化途径在向非光合组织提供丝氨酸方面起着重要作用。在大肠杆菌中过量生产的重组酶显示相对于3-磷酸甘油酸和NAD（+）的双曲线动力学。
In plants, Ser is synthesized through a couple of pathways. 3-Phosphoglycerate dehydrogenase (PGDH), the first enzyme that is involved in the phosphorylated pathway of Ser biosynthesis, is responsible for the oxidation of 3-phosphoglycerate to phosphohydroxypyruvate. Here we report the first molecular cloning and characterization of PGDH from Arabidopsis thaliana, Sequence analysis of cDNA and a genomic clone revealed that the PGDH gene is composed of three exons, encoding a 623-amino acid polypeptide (66,453 Da). The deduced protein, containing three of the most conserved regions in the NAD-dependent 2-hydroxyacid dehydrogenase family, has 38-39% identity to its animal and bacterial counterparts. The presence of an N-terminal signal sequence for translocation into plastids was confirmed by particle-gun bombardment experiments using green fluorescence protein as a reporter protein for subcellular localization. Southern hybridization analysis and restriction fragment length polymorphism mapping indicated that PGDH is a single-copy gene that is mapped to the upper arm of chromosome 1. Northern hybridization analysis indicated preferential expression of PGDH mRNA in root tissues of light-grown plants, suggesting that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying Ser to non-photosynthetic tissues. The recombinant enzyme overproduced in Escherichia coli displayed hyperbolic kinetics with respect to 3-phosphoglycerate and NAD(+).