Spinelli S L, Kierzek R, Turner D H, Phizicky E M
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
J Biol Chem. 1999 Jan 29;274(5):2637-44. doi: 10.1074/jbc.274.5.2637.
The last step of tRNA splicing in yeast is catalyzed by Tpt1 protein, which transfers the 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2"-cyclic phosphate (Appr>p). Structural and functional TPT1 homologs are found widely in eukaryotes and, surprisingly, also in Escherichia coli, which does not have this class of tRNA splicing. To understand the possible roles of the Tpt1 enzymes as well as the unusual use of NAD, the reaction mechanism of the E. coli homolog KptA was investigated. We show here that KptA protein removes the 2'-phosphate from RNA via an intermediate in which the phosphate is ADP-ribosylated followed by a presumed transesterification to release the RNA and generate Appr>p. The intermediate was characterized by analysis of its components and their linkages, using various labeled substrates and cofactors. Because the yeast and mouse Tpt1 proteins, like KptA protein, can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate, these enzymes likely use the same reaction mechanism. Step 1 of this reaction is strikingly similar to the ADP-ribosylation of proteins catalyzed by a number of bacterial toxins.
酵母中tRNA剪接的最后一步由Tpt1蛋白催化，该蛋白将连接的tRNA上的2'-磷酸转移至NAD，生成ADP-核糖1''-2''-环磷酸酯（Appr>p）。结构和功能上的TPT1同源物在真核生物中广泛存在，令人惊讶的是，在不具有此类tRNA剪接的大肠杆菌中也存在。为了了解Tpt1酶可能的作用以及NAD的异常用途，对大肠杆菌同源物KptA的反应机制进行了研究。我们在此表明，KptA蛋白通过一种中间体从RNA上去除2'-磷酸，在该中间体中，磷酸被ADP-核糖基化，随后可能发生转酯反应以释放RNA并生成Appr>p。使用各种标记的底物和辅因子，通过分析中间体的成分及其连接方式对该中间体进行了表征。由于酵母和小鼠的Tpt1蛋白与KptA蛋白一样，能够催化KptA生成的中间体转化为产物和原始底物，因此这些酶可能使用相同的反应机制。该反应的第一步与多种细菌毒素催化的蛋白质ADP-核糖基化极为相似。
