Smith P M, Mann A J, Goggin D E, Atkins C A
Department of Botany, The University of Western Australia, Nedlands.
Plant Mol Biol. 1998 Apr;36(6):811-20. doi: 10.1023/a:1005969830314.
A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.
从豇豆根瘤cDNA文库中分离出了一种编码磷酸核糖氨基咪唑（AIR）合成酶的cDNA（VUpur5），该酶是嘌呤从头生物合成途径中的第五种酶。它编码一个由388个氨基酸组成的蛋白质，预测分子量为40.4 kDa。推导的氨基酸序列与来自其他生物体的AIR合成酶具有显著同源性。AIR合成酶存在于豇豆根瘤的线粒体和质体中。VUpur5 cDNA编码的信号序列具有与质体转运序列相关的特性，但没有质体靶向蛋白预期的共有切割位点。尽管该信号序列不具有线粒体靶向蛋白的结构特征，但在成熟蛋白预测的N端附近有一个线粒体切割位点基序（RX/XS）。Southern分析表明AIR合成酶由单个基因编码，这引发了关于该基因产物如何靶向这两个细胞器的问题。与豇豆的其他组织相比，VUpur5在根瘤中的表达水平要高得多，并且该基因在固氮开始之前就已活跃。这些结果表明固氮产物在基因表达的初始诱导中不起作用。VUpur5在大肠杆菌中表达，并用重组蛋白制备抗体。这些抗体识别两种分子量不同的AIR合成酶形式。两种形式都存在于线粒体中，尽管较大的蛋白更为丰富。在质体中仅检测到较小的蛋白。
