Amblar Mónica, Arraiano Cecília M
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
FEBS J. 2005 Jan;272(2):363-74. doi: 10.1111/j.1742-4658.2004.04477.x.
Exoribonuclease II (RNase II), encoded by the rnb gene, is a ubiquitous enzyme that is responsible for 90% of the hydrolytic activity in Escherichia coli crude extracts. The E. coli strain SK4803, carrying the mutant allele rnb296, has been widely used in the study of the role of RNase II. We determined the DNA sequence of rnb296 and cloned this mutant gene in an expression vector. Only a point mutation in the coding sequence of the gene was detected, which results in the single substitution of aspartate 209 for asparagine. The mutant and the wild-type RNase II enzymes were purified, and their 3' to 5' exoribonucleolytic activity, as well as their RNA binding capability, were characterized. We also studied the metal dependency of the exoribonuclease activity of RNase II. The results obtained demonstrated that aspartate 209 is absolutely essential for RNA hydrolysis, but is not required for substrate binding. This is the first evidence of an acidic residue that is essential for the activity of RNase II-like enzymes. The possible involvement of this residue in metal binding at the active site of the enzyme is discussed. These results are particularly relevant at this time given that no structural or mutational analysis has been performed for any protein of the RNR family of exoribonucleases.
核糖核酸外切酶II（RNase II）由rnb基因编码，是一种普遍存在的酶，在大肠杆菌粗提物中90%的水解活性由其负责。携带突变等位基因rnb296的大肠杆菌菌株SK4803已被广泛用于研究RNase II的作用。我们测定了rnb296的DNA序列，并将这个突变基因克隆到一个表达载体中。在该基因的编码序列中仅检测到一个点突变，该突变导致天冬酰胺替代了天冬氨酸209。对突变型和野生型RNase II酶进行了纯化，并对它们从3'到5'的核糖核酸外切酶活性以及RNA结合能力进行了表征。我们还研究了RNase II外切核糖核酸酶活性对金属的依赖性。所得结果表明，天冬氨酸209对于RNA水解绝对必需，但对于底物结合并非必需。这是第一个证明酸性残基对于RNase II样酶活性至关重要的证据。讨论了该残基可能参与酶活性位点的金属结合。鉴于尚未对核糖核酸外切酶RNR家族的任何蛋白质进行结构或突变分析，此时这些结果尤其具有相关性。
