Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge T1K 3M4, Alberta, Canada.
J Phys Chem B. 2024 Oct 3;128(39):9455-9469. doi: 10.1021/acs.jpcb.4c05846. Epub 2024 Sep 23.
Human endonuclease V (EndoV) catalytically removes deaminated nucleobases by cleaving the phosphodiester bond as part of RNA metabolism. Despite being implicated in several diseases (cancers, cardiovascular diseases, and neurological disorders) and potentially being a useful tool in biotechnology, details of the human EndoV catalytic pathway remain unclear due to limited experimental information beyond a crystal structure of the apoenzyme and select mutational data. Since a mechanistic understanding is critical for further deciphering the central roles and expanding applications of human EndoV in medicine and biotechnology, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were used to unveil the atomistic details of the catalytic pathway. Due to controversies surrounding the number of metals required for nuclease activity, enzyme-substrate models with different numbers of active site metals and various metal-substrate binding configurations were built based on structural data for other nucleases. Subsequent MD simulations revealed the structure and stability of the human EndoV-substrate complex for a range of active site metal binding architectures. Four unique pathways were then characterized using QM/MM that vary in metal number (one versus two) and modes of substrate coordination [direct versus indirect (water-mediated)], with several mechanisms being fully consistent with experimental structural, kinetic, and mutational data for related nucleases, including members of the EndoV family. Beyond uncovering key roles for several active site amino acids (D240 and K155), our calculations highlight that while one metal is essential for human EndoV activity, the enzyme can benefit from using two metals due to the presence of two suitable metal binding sites. By directly comparing one- versus two-metal-mediated P-O bond cleavage reactions within the confines of the same active site, our work brings a fresh perspective to the "number of metals" controversy.
人类内切核酸酶 V（EndoV）通过切割磷酸二酯键，催化去除脱氨碱基，这是 RNA 代谢的一部分。尽管 EndoV 与多种疾病（癌症、心血管疾病和神经紊乱）有关，并可能成为生物技术中的有用工具，但由于除了 apoenzyme 的晶体结构和一些选择性突变数据之外，实验信息有限，因此人类 EndoV 催化途径的详细信息仍不清楚。由于对人类 EndoV 在医学和生物技术中的核心作用的进一步解读以及应用拓展的理解具有重要意义，因此使用分子动力学（MD）模拟和量子力学/分子力学（QM/MM）计算来揭示催化途径的原子细节。由于围绕核酶活性所需金属数量的争议，基于其他核酶的结构数据构建了具有不同数量活性位点金属和各种金属-底物结合构型的酶-底物模型。随后的 MD 模拟揭示了一系列活性位点金属结合结构下人类 EndoV-底物复合物的结构和稳定性。然后使用 QM/MM 对具有不同金属数量（一个与两个）和底物配位模式（直接与间接（水介导））的四种独特途径进行了特征描述，其中几种机制与相关核酶的实验结构、动力学和突变数据完全一致，包括 EndoV 家族的成员。除了揭示几个活性位点氨基酸（D240 和 K155）的关键作用外，我们的计算还强调，尽管一个金属对于人类 EndoV 活性是必需的，但由于存在两个合适的金属结合位点，该酶可以受益于使用两个金属。通过在相同的活性位点内直接比较一价和二价金属介导的 P-O 键断裂反应，我们的工作为“金属数量”争议带来了新的视角。
