The nondiscriminating aspartyl‐tRNA synthetase (ND‐AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNA^Asp and tRNA^Asn generating a correctly charged Asp‐tRNA^Asp and an erroneous Asp‐tRNA^Asn. This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon‐binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera‐D and Chimera‐N, by replacing the native anticodon‐binding domain in the Helicobacter pylori ND‐AspRS with that of a discriminating AspRS (Chimera‐D) and an asparaginyl‐tRNA synthetase (AsnRS, Chimera‐N), both from Escherichia coli . Both chimeric enzymes showed similar secondary structure compared to wild‐type (WT) ND‐AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera‐D was more discriminating as it aspartylated tRNA^Asp over tRNA^Asn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera‐N exhibited low catalytic activity toward tRNA^Asp and was unable to aspartylate tRNA^Asn. The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli . Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon‐binding domain and the catalytic domain in Chimera‐N compared to Chimera‐D or WT, explaining its lower stability and catalytic activity.

中文翻译：

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非区分性天冬氨酰-tRNA合成酶中的反密码子结合域交换显示出对tRNA特异性和催化活性的贡献。

在许多古细菌和细菌中发现的无差别的天冬氨酰tRNA合成酶（ND-AspRS）将天冬氨酸共价结合到tRNA ^Asp和tRNA ^Asn上，生成带正电荷的Asp-tRNA ^Asp和错误的Asp-tRNA ^Asn。这种松弛的tRNA特异性由tRNA和酶之间的相互作用控制。为了评估反密码子结合结构域对tRNA特异性的贡献，我们通过将幽门螺杆菌ND-AspRS中的天然反密码子结合结构域替换为a的结构，构建了两种嵌合酶Chimera-D和Chimera-N。判别ASPRS（嵌合体-d）和天冬酰胺酰-tRNA合成酶（AsnRS，嵌合体-N），都来自大肠杆菌。与野生型（WT）ND-AspRS相比，两种嵌合酶均显示出相似的二级结构，并保持了在溶液中形成二聚体复合物的能力。尽管Chimera-D的催化活性不如WT，但更具区分性，因为它将tRNA ^Asp相对于tRNA ^Asn进行了天冬氨酰化，特异性比为7.0，而WT酶为2.9。相反，Chimera-N对tRNA ^Asp表现出低催化活性，并且不能使tRNA ^Asn天冬^酰胺化。当在大肠杆菌中表达时，观察到的两种嵌合体的催化活性与其异源毒性相关。。分子动力学模拟显示，与Chimera-D或WT相比，Chimera-N中反密码子结合域和催化域之间的界面氢键网络减少，这说明其稳定性和催化活性较低。