The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel.
The kinase activity of the Helicobacter pylori Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase is sensitive to distal mutations in its putative ammonia tunnel.
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幽门螺杆菌 Asp-tRNA(Asn)/Glu-tRNA(Gln) 酰胺转移酶的激酶活性对其假定的氨通道中的远端突变敏感。
DOI：
10.1021/bi201143x
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Hendrickson,TamaraL
中科院分区：
文献类型：
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作者：
Zhao,Liangjun;Dewage,SajeewaW;Bell,MichaelJ;Chang,Keng-Ming;Fatma,Shirin;Joshi,Nilesh;Silva,Gayathri;Cisneros,GAndres;Hendrickson,TamaraL
幽门螺杆菌（Helicobacterpylori，Hp）Asp-tRNAAsn/Glu-tRNAGlamidotransferase（AdT）在间接氨酰化和翻译保真度中起重要作用。AdT在两个独立的亚基中有两个活性位点。动力学研究表明，域间通信发生在这些亚基之间，然而，这种机制还没有得到很好的理解。为了探索AdT中结构域之间的通讯，我们调整了一种测定方法并对其进行了优化，以从动力学角度表征HpAdT的激酶活性。该试验应用于分析一系列的点突变在整个推定的AdT氨隧道连接的两个活性位点的保守位置。鉴定了导致AdT激酶活性显著降低（降低55-75%）的几种突变。在Thr 149（GatB激酶活性位点远端37 bp处）和Lys 89（位于加塔和GatB的界面处）的突变对AdT的激酶活性是有害的，这表明这些突变破坏了两个活性位点之间的域间通信。对野生型AdT、Thr 149处的缬氨酸突变和Lys 89处的精氨酸突变的模型进行分子动力学模拟。野生型，T149 V和K89 R AdT模拟结果的比较揭示了可能参与连接两个活性位点的59个常见残基。
TheHelicobacter pylori(Hp) Asp-tRNAAsn/Glu-tRNAGlnamidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain–domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity ofHpAdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT’s kinase activity (reduced by 55–75%) were identified. Mutations at Thr149 (37 Å distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT’s kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.