Molecular dissection of arginyltransferases guided by similarity to bacterial peptidoglycan synthases
Molecular dissection of arginyltransferases guided by similarity to bacterial peptidoglycan synthases
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DOI：
10.1038/sj.embor.7400747
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发表时间：
2006-08-01
期刊：
影响因子：
7.7
通讯作者：
Kashina, Anna
中科院分区：
文献类型：
--
作者：
Rai, Reena;Mushegian, Arcady;Kashina, Anna
翻译后蛋白质精氨酸化是小鼠心血管发育和血管生成所必需的，由精氨酸基转移RNA-蛋白质转移酶ATE1介导，ATE1是一类功能保守但知之甚少的酶。在这里，我们利用序列分析来检测Ate1家族和细菌FemABX家族的氨酰-tRNA-肽转移酶之间的进化关系，并预测精氨酸基转移酶中重要的功能残基，然后利用这些残基构建一组突变体，为进一步对小鼠ATE1进行分子解剖。预测的功能重要区域中残基的点突变导致酶活性的变化，包括小鼠ATE1的完全失活；其他突变改变了其底物特异性。我们的结果首次揭示了ATE1介导的精氨酸转移反应和底物识别的机制，并定义了一个新的蛋白质超家族Dupli-gnat，通过复制GNAT乙酰转移酶结构域来反映其起源。
Post-translational protein arginylation is essential for cardiovascular development and angiogenesis in mice and is mediated by arginyl-transfer RNA-protein transferases Ate1-a functionally conserved but poorly understood class of enzymes. Here, we used sequence analysis to detect the evolutionary relationship between the Ate1 family and bacterial FemABX family of aminoacyl-tRNA-peptide transferases, and to predict the functionally important residues in arginyltransferases, which were then used to construct a panel of mutants for further molecular dissection of mouse Ate1. Point mutations of the residues in the predicted regions of functional importance resulted in changes in enzymatic activity, including complete inactivation of mouse Ate1; other mutations altered its substrate specificity. Our results provide the first insights into the mechanisms of Ate1-mediated arginyl transfer reaction and substrate recognition, and define a new protein superfamily called Dupli-GNAT to reflect its origin by the duplication of the GNAT acetyltransferase domain.