Dmd066829 227..237

The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones.CYP11A1 catalyzes the side-chain cleavageof cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of glucoand mineralocorticoids, respectively. This study reveals their additional capability tometabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17b-hydroxy-17a-methylandrosta-1,4dien-3-on),which is a commondoping agent. By contrast,microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 mM and 5.4 mM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged forCYP11A1.Catalytic efficienciesofOTconversion were determined to be 46min mM for CYP11A1, 741min mM for CYP11B1, and 3338 min mM for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolismby the CYP11B subfamily members were produced at amilligram scale with a recombinant Escherichia coli–based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11b-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11b,18-diOH-OT and 11b-OH-OT-18-al, which rearranges to its tautomeric form 11b,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography–tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies.