Since amino acids of sequence motif 1 line the nucleophile-binding pocket of HHDHs, we also expected a possible impact of those residues on nucleophile binding, as already observed for the nucleophile azide during our kinetic studies of motif 1 mutants. Thus, we further focused on a potential change in nucleophile acceptance after mutagenesis of motif 1 residues using a broader range of nucleophiles. In this regard, mutants T7S, F12Y and G14A of HheC as well as HheG mutants T13S, Y18F and G20A were applied in epoxide ring opening reactions of phenyl glycidyl ether (4) using azide, nitrite, cyanide, cyanate, thiocyanate as well as the halides chloride and bromide as nucleophiles. Those nucleophiles have previously been demonstrated to be accepted by HheC and HheG wild type. To cover an activity range as large as possible, conversions of enzyme-catalyzed transformations were determined after short (1 h) but also extended (24 h) reaction times (Figure ). These experiments revealed that the overall nucleophile acceptance of HheC and HheG was not altered considerably upon mutagenesis. However, a few interesting results stand out. For instance, HheG mutant G20A displayed surprisingly high activity with the nucleophiles thiocyanate and bromide, almost in the range of wild-type HheG, while it was virtually inactive with all other tested nucleophiles. However, the same effect was not noticed for HheC G14A, which might be related to the overall much lower activity of HheC with thiocyanate. As reported earlier , epoxide ring opening with thiocyanate can occur via S-and N-nucleophilic attack yielding two different product isomers, which were also observed in our study. Their ratio, however, did not change depending on the applied enzyme variant (Figure ). In contrast, the ratio of formed diol and nitroalcohol product in the ring opening of 4 with nitrite as nucleophile indeed varied to some extent depending on the respective motif 1 mutation. In this case, the diol product occurs due to O-nucleophilic attack at the epoxide and subsequent hydrolysis of the formed nitrite ester. Interestingly, especially the preference of HheC mutant T7S and HheG mutant G20A for diol formation was increased in comparison to respective wild-type enzymes (Figure ). Those results again underscore the impact of 1 residues on nucleophile binding and selectivity. ) with 20 mM nucleophile (azide, nitrite, cyanide, cyanate, thiocyanate, chloride, bromide) in 50 mM Tris•SO4 buffer, pH 7.0, at 30 °C (HheC) or 22 °C (HheG) and 900 rpm using each 150 µg mL -1 purified enzyme. Reactions were carried out in a total volume of 1 mL. Samples were taken after 1 h and 24 h, extracted with an equal volume of tert-butyl methyl ether and analyzed by achiral GC. "NC" represents negative control reactions without enzyme addition. B) Ratio of formed products in reactions with nucleophiles nitrite and thiocyanate.