Sahrawy M, Chueca A, Hermoso R, Lázaro J J, Gorgé J L
Department of Plant Biochemistry, Estación Experimental del Zaidín (CSIC), Granada, Spain.
J Mol Biol. 1997 Jun 20;269(4):623-30. doi: 10.1006/jmbi.1997.1054.
The alignment of the six higher plant photosynthetic fructose-1,6-bisphosphatases (FBPases) so far sequenced shows a lack of homology in the region which just precedes the cluster engaged in light modulation. Earlier experiments suggested that this region is the docking point in FBPase-thioredoxin (Trx) binding, and could be responsible for the interspecific differences in the enzyme-Trx interaction and Trx ability for FBPase activation. Using a pea chloroplast FBPase-coding cDNA, we have prepared two chimeric clones for FBPase. One of them (pDELFBP) shows a deletion of the 17 amino acids (Leu154 to Glu170) coding sequence, whereas in the second (pPFBPW) the above sequence was substituted by the corresponding one of the wheat enzyme. After Escherichia coli overexpression in pET-3d and later purification, both modified FBPases showed FBPase activity when determined under non-reducing conditions. However, only DELFBP lost the Trx f modulatory effect, indicating the important role played by this fragment in FBPase-Trx interaction and activity. Under these conditions the substituted PFBPW enzyme retains FBPase activity, even though clearly diminished. Superose 12 filtration experiments after preincubating the wild-type and modified FBPases with Trx f, showed the existence of an enzyme-Trx f binding with the wild-type and the substituted PFBPW, but not with the deleted DELFBP protein. Similarly, gradient PAGE under native conditions, followed by Western blot and developing with FBPase and Trx f antibodies, indicated the existence of such a binding between the wild-type and PFBPW, on the one hand, and both Trxs f and m, on the other, although never with the deleted DELFBP enzyme. These results show the central role played by the regulatory site preceding fragment of chloroplast FBPase in its binding with Trx. Computer-aided tridimensional models for the wild-type and modified FBPases are proposed.
到目前为止已测序的六种高等植物光合果糖-1,6-二磷酸酶（FBPases）的比对显示，在参与光调节的簇之前的区域缺乏同源性。早期实验表明，该区域是FBPase-硫氧还蛋白（Trx）结合的对接点，可能导致酶-Trx相互作用和Trx激活FBPase能力的种间差异。利用豌豆叶绿体FBPase编码cDNA，我们制备了两个FBPase嵌合克隆。其中一个（pDELFBP）显示编码序列缺失17个氨基酸（Leu154至Glu170），而在第二个（pPFBPW）中，上述序列被小麦酶的相应序列取代。在pET-3d中进行大肠杆菌过量表达并随后纯化后，两种修饰的FBPases在非还原条件下测定时均显示出FBPase活性。然而，只有DELFBP失去了Trx f调节作用，表明该片段在FBPase-Trx相互作用和活性中起重要作用。在这些条件下，取代的PFBPW酶保留了FBPase活性，尽管明显降低。在将野生型和修饰的FBPases与Trx f预孵育后进行的Superose 12过滤实验表明，野生型和取代的PFBPW存在酶-Trx f结合，但缺失的DELFBP蛋白不存在。同样，在天然条件下进行梯度PAGE，随后进行Western印迹并用FBPase和Trx f抗体显色，表明一方面野生型和PFBPW之间存在这种结合，另一方面Trxs f和m之间也存在这种结合，尽管从未与缺失的DELFBP酶结合。这些结果表明叶绿体FBPase调节位点前片段在其与Trx结合中起核心作用。提出了野生型和修饰的FBPases的计算机辅助三维模型。
