Efficient regio- and stereo-selective C-H bond hydroxylation of steroids using an engineered heme-thiolate peroxygenase biocatalyst

A crucial reaction in chemical synthesis is the activation of unactivated carbon-hydrogen bonds in complex molecules. We demonstrate the regio- and stereo-selective hydroxlation of the steroids progesterone and androstenedione using the peroxygenase activity of an engineered bacterial cytochrome P450 enzyme, CYP154C8. By replacing a single amino acid of the I-helix we change this monooxygenase enzyme into a peroxygenase, enabling the efficient and selective biocatalytic formation of the 16 alpha-hydroxy steroid metabolite. A steroid hydroxylating monooxygenase enzyme was converted into a peroxygenase to enable the stereoselective hydroxylation of C-H bonds in these complex molecules simply by adding hydrogen peroxide.