To investigate whether this enantioselectivity improvement of HheG holds true for other reactions as well, the fully randomized library at positions 44 to 46 was further screened in the azidolysis of styrene oxide (3) and trans-2,3-heptene oxide (5) as well as the ring-opening of cyclohexene oxide (1) with cyanide (Scheme S1 and Figure ). With cyanide as nucleophile, conversions of all HheG variants were much smaller compared to azide (Table ), in agreement with previous reports. Remarkably, variant M45F again displayed higher enantioselectivity, this time, however, for the opposite cyanoalcohol enantiomer compared to wild-type HheG 42 , as well as a three-times higher conversion (Table ). Also other aromatic residues at position 45 resulted in preferential formation of the (1S,2R)-product enantiomer, while HheG variants T44F and T44Y displayed slightly increased enantioselectivity compared to wild type for production of (1R,2S)-2-cyanocyclohexan-1-ol (Figure and Table ). Thus, an only minor difference in positioning of the aromatic residue on loop4947 determines whether one or the other product enantiomer is formed, at least in the conversion of 1 with cyanide. Inversion in enantioselectivity upon loop engineering has also been reported for other HHDHs, e.g. for HheC from Agrobacterium radiobacter AD1 and HheA from Arthrobacter sp. strain AD2 in the dehalogenation of 2-chloro-1-phenylethanol. Both enzymes, however, do not possess such a highly flexible loop close to the N-terminus that would correspond to loop3947 of HheG. With styrene oxide (3) as substrate, HheG preferentially catalyzes nucleophilic attack at the benzylic α-position (Scheme S1), which is the same as in the non-enzymatic reaction but opposite to the regioselectivity of most other HHDHs. Thus, to preclude a reduction in product enantiomeric excess during azidolysis of 3 due to chemical background, screening reactions with 3 and azide were already stopped after 10 min, attaining between 50 and 60% of conversion (Table ). This time, several loop variants displayed an increase in enantioselectivity with preferential formation of (S)-2-azidophenylethan-1-ol (4) like HheG wild type (Figure ), while highest E-values of 40 and 46 were observed for variants carrying a phenylalanine at position 44 or 45, respectively, corresponding roughly to a two-fold improvement in comparison to wildtype HheG (Table ). Hence, mutation M45F in HheG does not only impact the enzyme's enantioselectivity in the conversion of cyclohexene oxide, but also in the transformation of a structurally unrelated epoxide like styrene oxide. Azidolysis of the internal epoxide 5 by HheG results in the formation of two different regioisomers, 2-azidoheptan-3-ol (6a) and 3-azidoheptan-2-ol (6b) (Scheme S1). Both are roughly produced in equal amounts by wild-type HheG and this ratio did not significantly change upon loop engineering (Table ). For several HheG variants, however, an inversion in enantiopreference compared to wild-type enzyme for formation of regioisomer 6a could be observed (Figure ), even though absolute E values were still quite low (highest E value of 2.8 for HheG V46R) (Table ). In contrast, the enantiopreference of the loop variants in the formation of regioisomer 6b was the same as for HheG wild type (Figure ), but the enantioselectivity of many variants increased reaching eeP values of up to 70% (for comparison, the eeP of 6b in the wild-type control was only 12%) (Table ). This time, HheG variants T44K, M45L, M45K and V46R turned out to yield highest selectivity improvements, while HheG M45F displayed only minor changes in enantioselectivity compared to wild type (Figure and Tables ).