In summary, screening of the fully randomized library at positions 44 to 46 with different substrates and nucleophiles always revealed several amino acid exchanges exerting a strong impact on HheG's enantioselectivity. This confirms our initial hypothesis that more selective HheG variants can indeed be accessed through engineering of loop3947. Moreover, only one mutation (M45F) on loop3947 was sufficient to obtain a highly selective HheG mutant for the azidolysis of cyclohexene oxide (1), giving azidoalcohol (1S,2S)-2a with 96%ee. In contrast, in the work by Tian et al. based on the structure-guided mutagenesis of active-site residues of HheG, a triple mutant (Y18G/M189L/F200W) was necessary to obtain the same product enantiomer with 94%ee. This further emphasizes the importance of loop3947 for the enantioselectivity of HheG.