Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion mechanism, originally discovered by the Steitz T. A. & Steitz J. A. A. (Proc. Natl. Acad. Sci. USA 1993, 90, 6498).
Extensive structural studies have portrayed the two-metal-ion architecture across the three domains of life. However, X-ray crystallography alone cannot exhaustively unravel how the two ions execute their functional role along the enzymatic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states (TS). Multiscale molecular simulations have been shown to be effective in disclosing the mechanistic aspects of the two-metal-aided catalysis for two prototypical enzymatic targets for drug discovery – i.e., ribonuclease H (RNase H) & type II topoisomerase (topoII) (Palermo et al., Acc. Chem. Res. 2015, 48, 220, Palermo et al., JCTC, 2013, 9, 857).
We used Ab-initio molecular dynamics & free energy methods (i.e., thermodynamic integration & metadynamics) to describe the role of the cooperative motion of the Mg ions during the enzymatic catalysis, revealing the fundamental aspects of the DNA/RNA processing. Our work is voted at clarifying the functional differences in the phosphodiester bond cleavage in RNA & DNA.
By integrating existing structural data with Born-Oppenheimer MD and a Quantum Mechanics/Molecular Mechanics approach, we proposed a model for the yet uncharacterized structure of the reactant state of type II topoisomerase, a metalloenzyme targeted by clinical antibiotics and anticancer agents. This model describes a canonical two-metal-aided mechanism and suggests how the metals could rearrange at the catalytic pocket during enzymatic turnover, explaining also a number of experimental evidences for topoII inhibition.
An other interesting observation from our simulations is the presence of a third metal ion (MgC) close to the catalytic site. A conserved acidic residue (E/D) stably coordinates MgC, which freely access the active site, thanks to a negatively charged connecting channel from the bulk. This suggests a mechanism of metals uptake and release upon DNA cleavage, which might ensure the catalytic turnover.
These outcomes contribute in clarifying the metal-aided catalysis in enzymes processing RNA & DNA. Encouraged by the insights provided by computational approaches, we foresee further experiments that will feature the functional and joint dynamics of the catalytic metal ions for nucleic acid processing. This could impact the de novo design of artificial metallonucleases and the rational design of potent metal-chelating inhibitors of pharmaceutically relevant enzymes.