Gone Swapna, Alfonso-Prieto Mercedes, Paudyal Samridhdi, Nicholson Allen W
Department of Chemistry, Philadelphia PA, 19122, USA.
Institute for Computational Molecular Science, Philadelphia PA, 19122, USA.
Sci Rep. 2016 May 6;6:25448. doi: 10.1038/srep25448.
Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4-fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.
核糖核酸酶III（RNase III）是一种保守的、具有基因调控作用的细菌内切核酸酶，可切割多种编码和非编码RNA中的双螺旋结构。RNase III受到多种水平的调控，这反映了其全局调控功能。已知在噬菌体T7感染期间，大肠杆菌（Ec）RNase III的催化活性会增加，这反映了噬菌体编码的蛋白激酶T7PK的表达。然而，催化增强的机制尚不清楚。本研究表明，纯化的T7PK可在体外将Ec-RNase III的丝氨酸磷酸化，并确定靶点为N端催化结构域中的Ser33和Ser34。动力学实验表明，磷酸化后kcat增加5倍，Km降低1.4倍，催化效率提高7.4倍。磷酸化不会改变单周转条件下底物切割的速率，这表明磷酸化增强了产物释放，而产物释放也是稳态中的限速步骤。分子动力学模拟提供了一种促进产物释放的机制，其中Ser33磷酸单酯与Arg95胍基形成盐桥，从而削弱RNase III与产物的结合。模拟还表明了为什么在任一丝氨酸处进行谷氨酸替代都不会产生增强作用，从而突出了对磷酸单酯的特定要求。
