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.

Scalable and Stereoselective C–H Bond #Hydroxylation of Steroids Using a Designer #Heme-Thiolate #Peroxygenase Biocatalyst | The Journal of Organic Chemistry pubs.acs.org/doi/10.1021/...

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Microbial Adaptation to Biodegradable #Microplastics Drives Transformation of Dissolved Organic Matter in #Soil

doi.org/10.1021/acs....

#SoilOrganicMatter #agriculture #enzymes #soilbacteria #Environment #isotopes #reactomics, #hydroxylation

Using #P450 #peroxygenases for the alpha-hydroxylation of fatty acids, in up to 150 mM substrate concentration reaching crazy TONs! Congratulations to Klara and all coauthors for this impressive work! #biocatalysis #elkcrew #hydroxylation @green_rsc ...