Building on these precedents, we therefore set out to systematically engineer the conserved motif 1 residues in two representative HHDHs, HheC from A. radiobacter as well as HheG from Ilumatobacter coccineus. We selected these HHDHs based on their biocatalytic relevance, the fact that both enzymes offer highresolution crystal structures, as well as their highly dissimilar catalytic properties. HheC is the by far best studied member of the HHDH family, generally displaying high catalytic activity and enantioselectivity in the dehalogenation of various substrates as well as in the ring opening of terminal epoxides. Moreover, numerous protein engineering studies of HheC have been published aiming either at an increased activity, enantioselectivity or stability of the enzyme. In contrast, HheG is rather thermolabile and much less selective. However, this HHDH was the first reported enzyme with relevant ring-opening activity towards sterically more demanding internal epoxides (cyclic as well as acyclic). Even though less protein engineering data are available for HheG so far, the enzyme exhibits distinct structural differences compared to HheC. This includes a much broader and open active site, an additional α-helix in the nucleophile binding site loop, as well as a long and highly flexible loop in the N-terminal part of the protein, modulating substrate access to but possibly also substrate binding in the active site. In this work, we experimentally examined all possible single mutants of HheC and HheG with defined amino acid exchanges at the three conserved motif 1 residues, namely threonine, phenylalanine/tyrosine and glycine (Figure ), to investigate the impact of each residue on enzyme activity, selectivity and stability. In this regard, our in-depth characterization revealed HheC F12Y to be enhanced considerably and towards multiple parameters. Complementary molecular dynamics (MD) and quantum mechanics (QM) simulations highlighted the formation of additional hydrogen bonds in mutant F12Y compared to HheC wild type, resulting in a better preorganization of the active site and a lower activation barrier for the epoxide ring-opening reaction. In addition, a quantitative workflow for spectrophotometric activity determination in epoxide ring opening reactions has been developed, enabling also fast and reliable kinetic measurements.