Abstract
Selective activation of steroid skeletons is crucial for the generation of pharmaceutically valuable compounds. Despite the success of engineering cytochrome P450 enzymes as peroxygenases to utilize cost-effective H<sub>2</sub>O<sub>2</sub>, their applications are limited due to poor H<sub>2</sub>O<sub>2</sub> tolerance. Here, we report a cytochrome P450 enzyme, namely P450stri, from Streptomyces triculaminicus and its engineering as a peroxygenase with strong H<sub>2</sub>O<sub>2</sub> tolerance for regioselective hydroxylation of steroids. We find that a conserved Phe above heme cluster predominantly determines the performance of peroxygenase activity and regioselectivity. Additionally, we reconstruct P450stri by two-dimensional engineering approach based on a Round Flask model to simultaneously increase selectivity and activity, yielding in the most effective 15β steroid hydroxylase variant using H<sub>2</sub>O<sub>2</sub> as co-substrate. Moreover, we further transform the beneficial variants to corresponding residues in the members of "P450stri branch", converting several P450 monooxygenases to peroxygenases with improved activity and selectivity. The present study provides insights into rational switch of enzymatic function, thus shedding a light on P450 enzymes on their biocatalytic applications.