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

24 April 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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.


phosphodiester bond

Supplementary materials

Supplementary Information
Additional information for comparisons of mouse and apo-human EndoV structures and the human EndoV QM/MM model (Figure S1), starting models for MD simulations (Figure S2), RMSDs (Figure S3 and Table S1), MD snapshots used to build QM/MM models (Figure S4), QM regions of all QM/MM models (Figure S5), comparisons of QM/MM ICs for two-metal mediated mechanisms (Figure S6), chemical mechanisms and key structural parameters for QM/MM stationary points (Figures S7, S9, and S10), representative crystal structures of one- and two-metal dependent nucleases (Figure S8), comparisons of human EndoV QM/MM RCs and mouse EndoV crystal structure (Figure S11), and percent occupancies of reaction parameters and average metal coordination distances from MD simulations (Tables S2 and S3).


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