De Vivo Marco, Dal Peraro Matteo, Klein Michael L
Center for Molecular Modeling and Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA.
J Am Chem Soc. 2008 Aug 20;130(33):10955-62. doi: 10.1021/ja8005786. Epub 2008 Jul 29.
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 x 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 x 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 Mg(2+) 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 beta-phosphoglucomutase crystal (Lahiri, S. D.; et al. Science 2003, 299 (5615), 2067-2071). 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.
核糖核酸酶H（RNase H）属于核苷酸转移酶（NT）超家族，可水解RNA与DNA杂交体中构成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药物设计中一个非常有前景的靶点。来自嗜碱芽孢杆菌（Bh）的RNase H/RNA与DNA复合物的高分辨率晶体结构提供了结构信息，该结构表明形成催化活性复合物需要两个金属离子。在这里，我们使用基于经典力场和量子力学/分子力学的计算方法来模拟RNase H中的核苷酸转移反应，阐明金属离子的作用和亲核试剂（水与氢氧根离子）的性质。在催化过程中，两个金属离子协同作用，促进亲核试剂的形成，并稳定过渡态和离去基团。重要的是，两个Mg(2+)金属还支持在反应过程中形成亚稳的磷正烷中间体，这类似于仅在有争议的β-磷酸葡萄糖变位酶晶体中获得的磷正烷中间体结构（Lahiri, S. D.;等人，《科学》2003年，299(5615)，2067 - 2071）。亲核试剂的形成（即水的去质子化）可以在一个质子从水转移到过渡态中可裂解磷酸根后原位实现。这种质子转移实际上是由溶剂化水分子介导的。由于酶的双金属基序具有高度保守性，这些结果可能也适用于属于NT超家族的结构相似的酶。
