Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme
Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme
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DOI：
10.1023/a:1005884304303
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发表时间：
1997-11-01
影响因子：
5.1
通讯作者：
Cullimore, JV
中科院分区：
文献类型：
--
作者：
Carvalho, H;Sunkel, C;Cullimore, JV
我们克隆并测序了元胞紫花苜蓿胞质谷氨酰胺合成酶(GS)多肽(a和b)的cDNA。利用这两个DNA，我们制备了一个编码b的N-末端结构域和a的C-末端结构域的结构，以产生一个结构域互换的多肽，它应该组装成一个含有嵌合活性位点的酶。原生酶和结构域互换酶都在大肠杆菌中表达，在那里它们具有催化和生理活性，因为它们能够拯救glnA缺失突变体。表达的多肽大小正确，同工酶在离子交换层析上的表现与其天然同源物相似。我们发现纯化的酶的动力学性质以及几种假定的细胞效应器对其活性的调节略有不同。纯化的a和b同源八聚体在体外解离，然后再结合，表明亚基能够自组装，可能是随机的，形成异构体同工酶。此外，通过对根瘤、根、茎和托叶的GS同工酶的研究，发现植物中存在异构体同工酶。
We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effecters. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.