As our studied loop3947 variants of HheG did not exhibit sufficient thermal stability while being improved in terms of activity and selectivity, promising variants from this study, namely M45F, M45Y and M45W, were subsequently combined with mutations at position 123 of HheG. Previously, we reported that aromatic amino acids as well as glycine at position 123 resulted in significantly thermostable HheG variants. Moreover, some of these variants, especially T123F and T123G, displayed enhanced activity as well. Based on crystal structure analysis and MD simulations, a possible interaction between positions M45 and T123 was hypothesized. Thus, respective double mutants were generated using site-directed mutagenesis, produced, purified and tested in reactions with the same epoxides and nucleophiles as used for our single mutants before. As desired, resulting double mutants of HheG exhibited higher apparent melting temperatures (up to 12 K increase) in comparison to wild-type HheG (Table ). Additionally, some double mutants were even further improved in terms of activity. Thus, variant M45F-T123G displayed an almost 2 times higher specific activity (14.6 U mg -1 ) in the azidolysis of 3 compared to the best single variant T44F (Table ). Likewise, this variant achieved also much higher conversions in the ring opening of 1 with cyanide (93% after 24 h) and cyanate (99% after 24 h, Table ) in comparison to HheG M45F (see Table ). Apart from that, enantioselectivity of the loop variants was generally not further improved by combination with mutations at position 123 (Table ). Interestingly, however, the selectivity of some double mutants for formation of nitroalcohol 2c in comparison to diol 2f in the ring opening of 1 with nitrite was decreased (Table ).