Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation.
Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation.
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DOI：
10.1038/srep25448
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发表时间：
2016-05-06
影响因子：
4.6
通讯作者：
Nicholson AW
中科院分区：
文献类型：
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作者：
Gone S;Alfonso-Prieto M;Paudyal S;Nicholson AW
核糖核酸酶III （RNase III）是一种保守的、基因调控的细菌内切酶，在多种编码和非编码rna中切割双螺旋结构。RNase III受到多个层面的控制，反映了其全球调控功能。已知大肠杆菌（Ec） RNase III的催化活性在噬菌体T7感染期间增加，反映了噬菌体编码蛋白激酶T7PK的表达。然而，催化增强的机制尚不清楚。本研究表明，体外纯化的T7PK可使Ec-RNase III在丝氨酸上磷酸化，并在n端催化区域鉴定出靶点为Ser33和Ser34。动力学实验表明，磷酸化后kcat增加5倍，Km减少1.4倍，催化效率提高7.4倍。在单次翻转条件下，磷酸化不会改变底物的裂解速率，这表明磷酸化促进了产物的释放，这也是稳定状态下的限速步骤。分子动力学模拟提供了促进产物释放的机制，其中Ser33磷酸单酯与Arg95胍基形成盐桥，从而减弱产物的RNase III结合。模拟还显示了为什么谷氨酸在任何丝氨酸上的取代都不赋予增强作用，从而强调了对磷酸单酯的特殊要求。
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.