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

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.