Following the initial qualitative activity screen, we studied the epoxide ring-opening activity of active mutants with azide and various epoxides in more detail to gain further insights into the kinetic implications of motif 1 mutations on a broader range of substrates. Specifically, we used structurally diverse epoxides to cover relevant chemical space by employing epichlorohydrin (1), cyclohexene oxide (2), styrene oxide (3), phenyl glycidyl ether (4), limonene oxide (5), and trans-1-phenylpropylene oxide (6) (Figure ) and followed their conversion with our quantitative BTB-based pH assay. This analysis revealed that the introduction of mutations in motif 1 significantly impacted overall enzymatic activity (Figure ), even though the substrate spectra of the HheC and HheG mutants did not change compared to the wild type. Indeed, most mutants exhibited considerable reductions of their specific activities with all model substrates compared to the respective wild-type enzyme. The only variant in this panel which gained significant activity was HheC mutant F12Y, displaying a 3-to 5-fold increase in specific activity with 1 and 3. Otherwise, even very conservative mutations, as for instance HheC mutant G14A and HheG mutant G20A, resulted in drastic losses of activity. This observation is in general agreement with the report of Jörnvall et al. on the mutagenesis of the corresponding conserved glycine residue in motif 1 of an SDR enzyme. Replacement of this glycine by alanine resulted in a 69% decrease in activity, while mutations G14V and G14N yielded almost inactive enzymes. The activity trends observed for the epoxide ring-opening direction generally carried over to the dehalogenation catalyzed by the motif 1 mutants. We examined this with the haloalcohols 1,3-dichloro-2-propanol (1f), 2-chlorocyclohexanol (2f) and 1,3-dibromo-2-propanol (7g) (Figure ), whose conversion could be followed discontinuously with a previously reported halide release assay. While the specific activities of nearly all mutants were significantly reduced compared to the wild-type enzymes, HheC F12Y displayed considerably increased activity with the haloalcohols 1f and 7g (Figure ), mirroring the trends observed for the ring-opening activities. A higher activity of HheC mutant F12Y in the dehalogenation of 1,3-dichloro-2-propanol (1f) has previously been observed during thermostabilization of HheC by directed evolution. Reactions were performed in duplicate in a total volume of 1 mL with 10 mM epoxide and 20 mM azide in 2 mM MOPS buffer, pH 7.0 at 30 °C (HheC) or 22 °C (HheG) using 20-400 µg mL -1 purified enzyme (Table ). Samples were taken after 30, 60, 180, 270 and 360 s. Chemical background of negative control reactions without enzyme addition was subtracted. The resulting specific activities exhibit standard deviations between 0.00 and 0.13 U mg - 1 . C) Specific activities of HheC and HheG mutants as well as wild-type enzymes in the dehalogenation of haloalcohols 1,3-dichloro-2-propanol (1f), 2-chlorocyclohexanol (2f) and 1,3-dibromo-2-propanol (7g) determined via halide release assay. Reactions were carried out in duplicate in a total volume of 1 mL with 10 mM haloalcohol in 25 mM Tris•SO4 buffer, pH 7.0 at 30 °C (HheC) or 22 °C (HheG) using 10-400 µg mL -1 purified enzyme (Table ). Samples were taken after 30, 60, 180, 270, and 360 s. Chemical background of negative control reactions without enzyme addition was subtracted. The resulting specific activities exhibit standard deviations between 0.00 and 0.21 U mg -1 .