Mechanism of Nucleic Acid Phosphodiester Bond Cleavage by Human Endonuclease V: MD and QM/MM Calculations Reveal a Versatile Metal Dependence

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 apo-enzyme 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 active site 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.