Furthermore, HheG variants lacking either the complete loop3947 or parts of it were investigated to gain further insights regarding the impact of this loop on HheG performance. To this end, three defined variants were generated: HheG Del39-47 with complete deletion of loop3947, HheG Del44-46 where only residues T44, M45 and V46 were eliminated, and HheG Ins-DPAE in which loop3947 was replaced by a short linker containing residues aspartate, proline, alanine and glutamate. This short sequence is present at the corresponding position of loop3947 in an HheG homolog from Actinomycetota bacterium (the same protein was also reported from Acidimicrobiia bacterium) that was recently described. Deletion of all residues from position 39 to 47 resulted in complete inactivation of HheG, even though soluble, and thus folded, enzyme could still be obtained (data not shown). In contrast, elimination of residues T44, M45, V46 (HheG Del44-46) as well as exchange of loop3947 with the short linker sequence (HheG Ins-DPAE) yielded active variants displaying significantly increased specific activity in the azidolysis of 3 compared to wild-type HheG, while the activity in the ring opening of 1 was generally reduced (Table ). The enantioselectivity of both variants was also altered compared to HheG wild type, but no general trend could be observed (Table ). Interestingly, both deletion variants again formed (1S,2R)-2b with slight preference in the cyanolysis of 1, which is the opposite enantiomer to that formed by HheG wild type preferentially. This further underlines the importance of loop3947 for regulation of HheG's activity and enantioselectivity, probably through differences in substrate und nucleophile positioning within the active site. Remarkably, complete deletion of loop3947 (as in HheG Del39-47) considerably decreased enzyme stability as well, as evident from a decrease in apparent melting temperature (Tm) by 5 K in comparison to wild-type HheG. In contrast, corresponding Tm values of deletion variants Del44-47 and Ins-DPAE were only slightly reduced (Table ).