The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1)

Abstract

The steroid hydroxylation and carbon-carbon bond cleavage activities of cytochrome P450 17A1 (CYP17A1) are responsible for the production of glucocorticoids and androgens, respectively. The inhibition of androgen synthesis is an important strategy to treat androgen-dependent prostate cancer. We discuss the different enzymatic activities towards the various substrates of CYP17A1, demonstrating its promiscuity. Additionally, a novel interhelical interaction is proposed between the F-G loop and the B'-helix to explain the 16α-hydroxylase activity of human CYP17A1 with progesterone as the substrate. The techniques used by biochemists to study this important enzyme are also summarized. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.

Keywords: 17,20-Lyase; 17-Hydroxylase; Androgen; Cytochrome P450; Hypertension; Metabolic switching; Steroidogenesis.

Figure 1

The substrates and products of CYP17A1.

Figure 2

Strategy for deuterated compound synthesis, subjecting brominated precursors to zinc dust and deuterated acetic acid (AcOD).

Figure 3

¹H NMR spectra of deuterated progesterone analogs, which illustrate the labeled sites. The H-17 triplet (Panel C) collapses to a doublet with H-16α deuteration (Panel A, 19) and disappears with H-17 deuteration (Panel B, 20). The H-21 methyl singlet disappears with trideuteration at C21 (Panel C, 21). There is a trace amount of non-deuterated progesterone (H-17 proton at ~2.6 ppm) as shown in the NMR spectrum in Panel B, which can be integrated in order to determine the isotope abundance. CDCl₃ was used as the solvent and the spectra were referenced to the CHCl₃ singlet at 7.26 ppm.

Figure 4

(A) Hypothetical reaction coordinates for CYP17A1-catalyzed steroid 17- and 16α-hydroxylation reactions. The components in red illustrate the lowering of the zero point energy of the reacting bond when the deuterium label replaces the hydrogen at H-17. (B) HPLC UV-chromatograms, absorbance at 254 nm of products from CYP17A1 incubation with progesterone and the deuterated analogs, indicating metabolic switching (Fig. 3). Incubation conditions are described in reference [20].

Figure 5

Intramolecular KIE values can be determined by comparing the relative product distributions for the (A) natural abundance and the (B) deuterated substrates. For simplification purposes, the 16β- and 21- hydrogen abstraction steps are ignored. This figure illustrates the major difference in the rate of hydroxylation between (A) and (B) arises from the C-H abstraction steps (k_8H, k_8D, k_12H, k_12D), and these differences give rise to metabolic switching.

Figure 6

Exploded view of CYP17A1 crystal structure with bound abiraterone. MacPymol software was used to visualize the structure. Alanine-105 residue interacts with leucine-205 of the F-helix. Heme and abiraterone = dark blue, B′-helix = light blue, I-helix = yellow, F-G loop = green. PDB: 3RUK.

Figure 7

CYP17A1 mutation A105L. The introduction of leucine at residue 105 positions the substrate progesterone so that the 17- and 21- carbon positions are more exposed to the active oxoiron species (compound I) compared to the 16-position.

Figure 8

CYP17A1 hydroxylation at C-16, C-17, and C-21, illustrating the abstraction of 4 non-equivalent hydrogen atoms among these reactions. The equilibrium arrows following the oxygen activation step indicate the possibility of metabolic switching if one of the reactive C-H sites (16α-, 17-, 21-) is deuterated.

Figure 9

Different plausible mechanisms for C17-C20 bond cleavage for CYP17A1 of 17-hydroxypregnenolone (2) to form DHEA (3). (A): Ferric peroxide mechanism. (B–D): Compound I mechanism. “*O” = “¹⁸O”

Figure 10

Mechanism for andiene (5) synthesis from C21-steroid precursor (1) as proposed by Akhtar et al. from deuterated substrate [33] (A) and compound I proposals (B) and (C). “*O” = “¹⁸O”

Figure 11

Mechanistic proposals for the formation of androsta-5-en-3β,17α-diol (4) from pregnenolone (1) as shown by Akhtar et al. [33, 35] (A and B). (C) is an alternative compound I mechanism from the gem-diol intermediate of pregnenolone. Radical intermediate 1-r explains the 17α-stereochemistry of compound 4. “*O” = “¹⁸O”