Overall, we have demonstrated that loop3947 is highly important for regulating the activity as well as enantioselectivity of HheG. In this context, variant M45F was identified displaying greatly increased activity and improved enantioselectivity in the ring opening of cyclohexene oxide with various nucleophiles. Likewise, HheG T44F displayed the highest activity and enantioselectivity increase in the azidolysis of styrene oxide among the screened loop3947 variants. In contrast, complete deletion of this dynamic loop resulted in soluble but inactive enzyme. This highlights the significance of loop3947 for catalytic performance of HheG. As close homologs of HheG, which have recently been described by us, feature comparable loops, a similar impact of loop mutations on their catalytic performance can be expected. Hence, future protein enginering campaigns of HheG or its homologs to improve or alter activity and enantioselectivity should not only focus on active-site mutations but include loop variations as well.