Unveiling the crucial intermediates in androgen production

Abstract

Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17-C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.

Keywords: cytochrome P450; nanodiscs; peroxo-hemiacetal; resonance Raman spectroscopy; steroids.

Fig. 1.

Proposed pathway for biosynthesis of androstenedione and DHEA catalyzed by human CYP17A1 (4, 5).

Fig. 2.

Cytochrome P450 enzymatic cycle and formation of a peroxo-hemiacetal intermediate.

Fig. S1.

Model of CYP17A1 incorporated into a nanodisc. The cytochrome P450 molecule is shown as a green cartoon representation, with heme presented in red sticks. The phospholipid bilayer is shown in orange with oxygen atoms in red, and the scaffold protein encompassing the lipid bilayer is shown as a blue cartoon representation.

Fig. 3.

Thermal annealing of peroxo-ferric intermediates monitored by optical absorption spectroscopy in CYP17A1 with different substrates PREG (A) and 17-OH PREG (B). Shown are difference spectra obtained by subtracting the spectrum at 160 K from the spectra measured at temperatures gradually increasing from 161 K (baseline) to 185 K (maximal difference).

Fig. 4.

rR spectral data for irradiated dioxygen adducts of CYP17A1. All spectra were measured with a 442-nm excitation line at 77 K, and the total collection time of each spectrum was 6 h. The rR ¹⁶O₂-¹⁸O₂ difference traces in H₂O and D₂O buffers of irradiated oxy-CYP17A1 samples (before annealing) with PREG (A) and 17-OH PREG (B) and corresponding samples after annealing to 165 K (C) and (D) are shown. Using the isolated bands for the ¹⁶O-peroxo (802 cm⁻¹) and ¹⁸O-hydroperoxo (738 cm⁻¹) species, the I₇₃₈/I₈₀₂ increases from 0.72 to 1.42. Similar values were obtained using the data from samples prepared with D₂O buffers (from 0.77 to 1.59). Ann., annealed; Irr., irradiated.

Fig. S2.

Low-frequency rR spectra of irradiated (IRR) oxy-ND/CYP17A1 samples with PREG, ¹⁶O₂/H₂O (A), ¹⁸O₂/H₂O (B), ¹⁶O₂/D₂O (C), ¹⁸O₂/D₂O (D), and their difference traces are indicated. Spectra were measured with a 442-nm excitation line at 77 K, and the total collection time of each spectrum was 6 h. The modes associated with hydroperoxo-species exhibit a 5 to 3 cm⁻¹ downshift in D₂O buffer. The percentages of peroxo- and hydroperoxo- species are approximately equal. The difference traces were obtained by subtracting the ¹⁸O₂ spectrum from the ¹⁶O₂ spectrum in H₂O (Upper) or D₂O (Lower) buffer. Then, the absolute spectra and difference traces were corrected using linear functions in the regions where the oxygen-sensitive modes are present.

Fig. S3.

Low-frequency rR spectra of irradiated oxy-ND/CYP17A1 samples with 17OH-PREG, ¹⁶O₂/H₂O (A), ¹⁸O₂/H₂O (B), ¹⁶O₂/D₂O (C), ¹⁸O₂/D₂O (D), and their difference traces are indicated. Spectra were measured with a 442-nm excitation line at 77 K, and the total collection time of each spectrum was 6 h. There is only one ν(O-O) mode and one ν(Fe-O) mode that do not have H/D sensitivity and are assigned to the peroxo-species.

Fig. S4.

Low-frequency rR spectra of irradiated and annealed (Ann.) at 165 K oxy-ND/CYP17A1 samples with PREG, ¹⁶O₂/H₂O (A), ¹⁸O₂/H₂O (B), ¹⁶O₂/D₂O (C), ¹⁸O₂/D₂O (D), and their difference traces. Spectra were measured with a 442-nm excitation line at 77 K, and the total collection time of each spectrum was 6 h. The intensity ratio of ν(O-O) of hydroperoxo-species to ν(O-O) of peroxo-species is ∼2:1.

Fig. S5.

Low-frequency rR spectra of irradiated and annealed at 165 K oxy-ND/CYP17A1 samples with 17OH-PREG, ¹⁶O₂/H₂O (A), ¹⁸O₂/H₂O (B), ¹⁶O₂/D₂O (C), ¹⁸O₂/D₂O (D), and their difference traces. Spectra were measured with a 442-nm excitation line at 77 K, with the total collection time of each spectrum being 6 h. No new species were detected that could be assigned to either the acylperoxo- or hydroperoxo-form. The approximate loss of intensity of modes associated with the Fe-O-O fragment is 40–50% as judged by comparison with the ν₇ mode at 674 cm⁻¹, which serves as an internal standard.

Fig. 5.

rR spectral data for irradiated dioxygen adducts of CYP17A1 samples with 17-OH PREG annealed at 190 K. ¹⁶O₂/H₂O (A), ¹⁸O₂/H₂O (B), ¹⁶O₂/D₂O (C), ¹⁸O₂/D₂O (D), and their ¹⁶O₂-¹⁸O₂ difference traces. (D, Inset) Difference trace of scrambled oxygen (SC) and the ¹⁶O₂ spectrum. Spectra were measured with a 406-nm excitation line at 77 K, and the total collection time of each spectrum was 8–9 h.

Fig. S6.

¹⁶O₂-¹⁸O₂ difference traces of oxy-ND/CYP17A1 samples with 17OH-PREG irradiated and annealed at 165 K in H₂O buffer (A), in D₂O buffer (B), annealed at 190 K in H₂O buffer (C), and in D₂O buffer (D). Spectra were measured with a 413.1-nm excitation line at 77 K. The total collection time of each spectrum was 6–8 h. The ν(O-O) modes of residual ferrous dioxygen adducts at 1,135 cm⁻¹ and 1,070 cm⁻¹ were used as internal standards to evaluate the intensity increase of the new oxygen-sensitive species at around 790 cm⁻¹.

Fig. S7.

rR spectra of irradiated and annealed at 190 K oxy-ND/CYP17A1 samples with PREG, ¹⁶O₂/H₂O (A), ¹⁸O₂/H₂O (B), and their difference traces. Spectra were measured with a 406-nm excitation line at 77 K. The total collection time of each spectrum was 4 h. The spectra show only the presence of ν(O-O) modes of the residual precursor dioxygen adducts. In contrast to the case with 17OH-PREG–bound samples (Fig. 5), which displayed a 791-cm⁻¹ mode of the peroxo-hemiacetal intermediate, and whose intensity was comparable to the intensity of the residual dioxygen adduct, there are no oxygen-sensitive modes observed near 790 cm⁻¹ in the difference trace.

Fig. S8.

rR spectra of irradiated and annealed at 190 K oxy-ND/CYP17A1 samples with 17OH-PREG, ¹⁶O₂/H₂O (A), ¹⁶O¹⁸O (B), and their difference traces. Spectra were measured with a 406-nm excitation line at 77 K. The total collection time of each spectrum was 8 h. SC, scrambled oxygen.