Combining enhanced sampling molecular dynamics simulations with dynamic QM/MM-MD exploration of the mechanisms, we shed light at the molecular level on the maturation process of 5S rRNA in B. subtilis, performed by the M5 enzyme. Providing a small displacement of the scissile phosphate and the use of adequate force fields, the structure of the experimentally reconstructed complex is stable, with small conformational fluctuations of the interface. Enhanced sampling is critical to reveal the full extent of the conformational variability of the rRNA:M5 interface, which we show critically impacts the active site structure by modulating the distance and orientation between key reactive residues. This rich conformational landscape thus suggests a variety of possible reaction mechanisms, that depend on the active site pre-organization. Comprehensive characterization of the different possible mechanisms from each conformational basin suggests that the most favorable reaction route would involve deprotonation of the nucleophilic water by the active site Glu96 and reprotonation of the 3 ′ leaving group by a Mg 2+ -bound water. Contrary to other RNases, M5 activity does not rely on a bifurcated aspartate between the 2 active site cations, nor on the release of initially frustrated hydration shells.