Split leucine-specific domain of leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus.
Split leucine-specific domain of leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus.
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DOI：
10.1021/bi061026r
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发表时间：
2006-11
期刊：
影响因子：
2.9
通讯作者：
Jing-Jing Ma-Jing;Ming-Wei Zhao;E. Wang
中科院分区：
文献类型：
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作者：
Jing-Jing Ma-Jing;Ming-Wei Zhao;E. Wang
Aquifex aeolicus的亮氨酰-tRNA合成酶(Leucyl-tRNA Synthetase，LEURS)是目前已知的唯一一种异二聚体合成酶。它被命名为Leur Alphabeta；，其α和β亚基分别含有634和289个残基。与嗜热性嗜热菌一样，Leur Alphabeta也有一个很大的额外结构域，即插入催化结构域的亮氨酸专一性结构域。亚基分裂位点正好位于亮氨酸专一性结构域的中间，可能具有独特的功能。在此，我们构建并纯化了一系列由突变的α亚基和野生型β亚基或野生型的α亚基和突变的β亚基组成的Leurαβ突变体。测定了突变体的ATP-PPI交换和氨基酰化活性，以及突变体对小螺旋(Leu)的充电能力。用凝胶位移法检测突变体与tRNA的相互作用。位于α亚基区域的两个由8个和9个氨基酸残基组成的多肽被发现是该酶活性所必需的。我们还表明，Leur Alphabeta中的结构域在微型螺旋(Leu)识别中起着重要作用。此外，该结构域对异源二聚体的组装几乎没有影响，对整个酶的热稳定性起到了作用，并以预测的方式与同源tRNA相互作用。
Leucyl-tRNA synthetase (LeuRS) from Aquifex aeolicus is the only known heterodimer synthetase. It is named LeuRS alphabeta;, and its alpha and beta subunits contain 634 and 289 residues, respectively. Like Thermus thermophilus LeuRS, LeuRS alphabeta has a large extra domain, the leucine-specific domain, inserted into the catalytic domain. The subunit split site is exactly in the middle of the leucine-specific domain and may have a unique function. Here, a series of mutants of LeuRS alphabeta consisting of either mutated alpha subunits and wild-type beta subunits or wild-type alpha subunits and mutated beta subunits were constructed and purified. ATP-PPi exchange and aminoacylation activities and the ability of the mutants to charge minihelix(Leu) were assayed. Interaction of the mutants with the tRNA was assessed by gel shift. Two peptides of eight and nine amino acid residues in the domain located in the alpha subunit were found to be essential for the enzyme's activity. We also showed that the domain in LeuRS alphabeta plays an important role in minihelix(Leu) recognition. Additionally, the domain was found to have little impact on the assembly of the heterodimer, to play a role in the thermal stability of the whole enzyme, and to interact with the cognate tRNA in the predicted manner.