Phosphodiester cleavage in ribonuclease H occurs via an associative two-metal-aided catalytic mechanism.
Phosphodiester cleavage in ribonuclease H occurs via an associative two-metal-aided catalytic mechanism.
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DOI：
10.1021/ja8005786
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发表时间：
2008-08-20
影响因子：
15
通讯作者：
Klein ML
中科院分区：
文献类型：
--
作者：
De Vivo M;Dal Peraro M;Klein ML
核糖核酸酶H（RNase H）属于核苷酸转移酶（NT）超家族，水解形成RNA·DNA杂交体中RNA链骨架的磷酸二酯键。这种酶与复制起始和DNA拓扑结构恢复有关，是抗HIV药物设计的一个非常有前途的靶点。由耐盐芽孢杆菌（Bacillus halodurans，Bh）RNase H/RNA·DNA复合物的高分辨晶体结构提供了结构信息，揭示了形成催化活性复合物需要两个金属离子。在这里，我们使用经典的基于力场和量子力学/分子力学的计算来模拟RNase H中的核苷酸转移反应，澄清金属离子的作用和亲核试剂的性质（水与氢氧根离子）。在催化过程中，两种金属离子协同作用，促进亲核试剂的形成，稳定过渡态和离去基团。重要的是，两种Mg 2+金属还支持沿反应形成亚稳正膦中间体沿着，其类似于仅在有争议的β-磷酸葡萄糖变位酶晶体中获得的正膦中间体结构。亲核试剂的形成（即，水去质子化）可以在一个质子从水迁移到处于过渡态的易断裂磷酸盐之后原位实现。这种质子转移实际上是由溶剂化水分子介导的。由于酶促G450基序的高度保守性，这些结果也可能与属于NT超家族的结构相似的酶有关。
Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA·DNA hybrids. This enzyme is implicated in replication initiation and DNA topology restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resolution crystal structures of the complex RNase H/RNA·DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/molecular mechanics calculations for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water versus hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg2+ metals also support the formation of a meta-stable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated β-phosphoglucomutase crystal. The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water molecules. Due to the highly conserved nature of the enzymatic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily.
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影响因子：
3
作者：
Dal Peraro, M. D.;Spiegel, Katrin;Klein, Michael L.
通讯作者：
Klein, Michael L.
影响因子：
6.8
作者：
Dal Peraro, Matteo;Ruggerone, Paolo;Carloni, Paolo
通讯作者：
Carloni, Paolo
影响因子：
4.3
作者：
De Vivo, Marco;Cavalli, Andrea;Recanatini, Maurizio
通讯作者：
Recanatini, Maurizio
影响因子：
3.3
作者：
Ivanov, I;Chen, B;Klein, ML
通讯作者：
Klein, ML
影响因子：
4.1
作者：
Kalé, L;Skeel, R;Schulten, K
通讯作者：
Schulten, K