Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts
Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts
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DOI：
10.1007/bf00019099
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发表时间：
1996-11-01
影响因子：
5.1
通讯作者：
Martin, W
中科院分区：
文献类型：
--
作者：
Flechner, A;Dressen, U;Martin, W
通过消减差异杂交从两色Sorcumbicolor中分离编码卡尔文循环酶转酮醇酶（TKL; EC 2.2.1.1）的cDNA，并用于从菠菜中分离该酶的几个全长cDNA克隆。在噬菌体T-7启动子的控制下，诱导过表达菠菜TKL亚基的大肠杆菌细胞后，TKL活性增加8.6倍，显示了所编码的成熟亚基的功能特性。叶绿体定位的克隆酶显示在体外合成的前体摄取后，由分离的叶绿体加工。Southern印迹分析表明，TKL是由菠菜基因组中的单个基因编码的。TKL蛋白的高等植物叶绿体和非光合真核生物的细胞质被发现是出乎意料的相似真细菌的同源物，这表明这些核基因可能真细菌的起源。叶绿体TKL是已被克隆的最后一个明显定位于叶绿体的卡尔文循环酶，从而完成了高等植物中该途径所有酶的基因探针的分离。
A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T-7 promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.