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 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.