The RNase H topology is shared between many catalytic cores of enzymes that process nucleic acids. We therefore strategically mined the protein data bank for similar catalytic motifs to obtain a picture of the generality of the catalytic mechanism using the histidine and a third ion in cleaving nucleic acids. We used VMD to automatically select structures based on certain criteria. As criteria we used the presence of a histidine within 5 of two Mg 2+ , Ca 2+ or Mn 2+ ions in all structures with resolution better than 2.5 Å and manually inspected the resulting structures. From our analysis, we could determine that indeed catalytic sites of various nucleases are similarly composed of glutamates, aspartates and histidines as has been described before, but we were able to further reason the positioning of some of the residues based on the results presented in this article and the recently available in crystallo study of the reaction 10 as well as the QM/MM dynamics of the Cas9 active site. All of the identified enzymes belong to the RNHL superfamily. While the DEDD motif is relatively common among nucleases , we identified three different subclasses of this catalytic motif with histidine based on the PDB mining. Two with Glu bound to metal B (RNase H-like, RuvC-like, Figure ) and the other with Glu bound to metal A (RNase T-like, Figure ). Both the RNase H-like motif as well as the RNase T-like motif have been commonly referred to as DEDD motifs. The RuvC-like motif is referred to as DDE motif. For the RNase T-like structures, Zuo and Deutscher 25 established the DEDDh nomenclature to differentiate DEDD nucleases that have histidine from those with a tyrosine (DEDDy) in their active site. It is well known that the catalytic mechanism of these nucleases is strongly related to the coordination of the catalytic magnesium ions , therefore the RNase H-like and RNase T-like motifs are not the same DEDDh motif even though the position of the histidine is very similar (Figure ). In both types, the histidine is placed on a loop segment coordinating the nucleophile (Figure ). Lys/Arg is present which we consider to be atypical for a general RNase H but present in Bh RNase H (K196). MgA is coordinated by a backbone (bb) oxygen. Example PDBs: 4LD0, 4EP4, 4UN3, 1HJR, 3HVR Differences in the catalytic mechanism are mainly associated with MgB, which is penta-coordinated in the RNase H-type and hexa-coordinated in the RNase T-like, most likely due to different specificities for DNA and RNA substrates. In contrast in the RNase T-like enzymes MgA is penta-coordinated. For the first two types, the pKa of the glutamate is high (pKa 7.5-9 as calculated with PROPKA 3.1, see Table ) due to the relatively nonpolar micro environment around the pocket with the protonated oxygen. The high pKa for the active site Glu supports the role of the glutamate as proton donor (RNase H-like, RuvC like) or in stabilizing the WATnuc (RNase T-like) as it is significantly more basic than a standard Glu (pKa ~ 4.5). In QM/MM simulations of the mechanism of the -subunit of DNA pol III it was shown that the Glu in the RNase T-like motif can pick up the second proton from the WATnuc after cleavage of the scissile bond. We show that in the RNase H-like enzymes protonation occurs via the Glu coordinated to MgB. However, this pathway is not possible in the canonical DEDDh motif in the RNase T-like motif due to the different position of the Glu (Figure ). In the RNase T-like enzymes, there is an Asp exclusively coordinated to MgB via a water molecule but its pKa likely is much closer to a standard Asp (pKa ~3, see Table ), making protonation via this residue unlikely. The proton for the protonation of the leaving group thus likely originates from any of the up to three water molecules of the first solvation shell of MgB. For example in the RNase T structure solved by Hsiao et. al. MgB is solvated with 3 water ligands as opposed to zero water ligands in the RNase H-like and in the RuvC-like structures. This mechanism is also consistent with the lowering of pKa of Mg associated water molecules. In the QM/MM simulation of the RNase T-like -subunit of the pol III the protonation of the leaving O3' remained elusive, but pathways via the scissile phosphate were ruled out based on the calculated energy barriers of >30 kcal mol -1 . Thus, the protonation via the solvent ligands in the RNase T-like motif is the only viable alternative. One of the waters in the RNase T-like motif is in a similar position to the glutamate in the RNase H-like enzymes. In the RuvC-like enzymes the DEDD motif is not observed (only DDE) and the last Asp of the DEDD motif is possibly replaced by a His (e.g in S. pyogenes (Spy) Cas9 RuvC H983). This is supported by the fact that H→D mutation in Tth RuvC is shown to activate the enzyme, which would reconstitute the DEDD motif of RNase H. This indicates the strong similarity of the RuvC-type and the RNase H-type. In contrast to this finding, other studies report that the affinity of nitrogen (e.g., in histidine or imino side chains of nucleic acids) for Mg 2+ is low and in the QM/MM ab initio MD of the Cas9 active site it can be seen, that the stable distance of H983 to MgA is around 4.2 Å, which is not directly coordinating. We can reproduce the same distance if we align our QM/MM reactant structure to the inactive Spy Cas9 (PDB 4UN3) RuvC active site (Figure ). In the H→D mutation for Tth RuvC most likely the adjacent H982, which is also conserved, can act as a replacement general base.