Hybrid QM/MM-MD simulations were used to investigate the phosphate cleavage reaction mechanism by M5. All QM/MM-MD calculations were carried out with the sander module of the Amber23 software, 46,47 using a 1 fs time step. First explorations of different possible mechanisms were conducted using the semi-empirical DFTB3 48 method for the QM region with OPhyd 49 parameters, specifically developed to describe phosphorus chemistry with improved accuracy. The most promising mechanism was then re-examined (in progress) with a higher DFT level of theory, B3LYP/6-31G * , 50-52 using the Gaussian16/sander QM/MM interface. This functional and basis set were chosen as a good compromise between computational cost and proven accuracy in similar phosphoryl transfer or phosphate hydrolysis reactions. Dispersion corrections were recently shown to be detrimental to the description of the interaction of divalent cations with their surrounding, 55 and were thus not used here, where Mg 2+ ions are central for the reactivity. A 15 Å cutoff was set for QM/MM interactions.