Scalable and Stereoselective C-H Bond Hydroxylation of Steroids Using a Designer Heme-Thiolate Peroxygenase Biocatalyst

ABSTRACT

A challenging yet critical reaction in chemical synthesis is the selective activation of unactivated carbon-hydrogen bonds in complex molecules. Here we demonstrate the biocatalytic regio- and stereoselective hydroxylation of steroids, including progesterone, testosterone, adrenosterone, androstenedione, and dehydroepiandrosterone (DHEA), using a variant of an engineered bacterial cytochrome P450 enzyme, CYP154C35 from Nocardia otitidiscaviarum. We replaced two amino acid residues within the oxygen-binding groove of the I-helix, and this modification of the acid-alcohol pair to a glutamine and a glutamate set of residues had the effect of converting the enzyme into an efficient peroxygenase (CYP154C35QE). This enzyme performed the biocatalytic oxidation of the steroids stereoselectively to form the 16α-hydroxylated metabolites. In the presence of a substrate, CYP154C35QE was stable to relatively high concentrations of H2O2 but could function catalytically using lower concentrations. The enzyme tolerated 20% DMSO and was not denatured upon being preheated for 1 h at 40 °C. The enzyme was capable of high turnover numbers (∼6500), resulting in complete conversion of the substrate, enabling the formation, purification, and characterization of the enantiopure 16α-hydroxylated steroids at up to 120 mg scale.