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 C 17 -C 20 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 C 20 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.T he excessive production of androgen, which effectively fuels the progression of cancer, especially prostate cancer, was first treated by surgical methods (1), whereas more modern approaches are focused on the discovery and development of pharmaceuticals that can selectively inhibit androgen synthesis (2, 3). A member of the cytochrome P450 superfamily (4, 5), cytochrome P450 17A1 (CYP17A1), occupies a central role in the biosynthesis of steroid hormones in humans. As was first reported by Nakajin and Hall (6) and Nakajin et al. (7) for CYP17 from pig testis, this enzyme catalyzes two fundamentally different types of chemical transformations (5-11), with the first being the efficient hydroxylation of both of its primary substrates, pregnenolone (PREG) and progesterone (PROG), to 17-hydroxypregnenolone (17-OH) PREG and 17-hydroxypregnenolone, respectively (Fig. 1). Importantly, 17-OH PREG is further processed in a second step in which CYP17A1 now catalyzes, not another hydroxylation reaction, but a complex 17,20 carbon-carbon (C-C) bond cleavage (lyase) reaction. This step converts 17-OH PREG to dehydroepiandrosterone (DHEA), a process that represents a critical branch point in human steroidogenesis by providing the essential precursor to androgens and various corticosteroids (5-9). Although a similar C-C bond cleavage of 17-OH PROG is also mediated by CYP17A1, it is of less importance in humans because its efficiency is only about 2% of the efficiency of the physiologically important lyase reaction involving 17-OH PREG (10). Recognizing the intensive efforts currently underway to design and test substances that selectively inhibit CYP17A1 (2, 3), there is a pressing need to enhance our understanding of the relevant reaction mechanisms, a task that typically entails identification of key reaction intermediates. Dir...

Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis

“…Clearly, Mn- b 5 binds to CYP17A1, but is incapable of enhancing the lyase activity, the latter being enhanced if the native iron containing cyt b 5 is bound. Taken together, these experiments provide strong evidence that cyt b5 is serving as a redox partner in CYP17A1 catalytic cycle, most likely delivering a second electron to the oxy-complex and facilitating formation of the peroxo-ferric complex, which recent evidence shows to be the catalytically active intermediate in the lyase reaction [35]. This result is in agreement with conclusions on the role of cyt b 5 in CYP2B4 catalysis [14,36].…”

“…Importantly, presence of cyt b 5 increases the coupling efficiency about 5-fold. Given the relatively high redox potential of cyt b 5 (~0 mV NHE) [4], its redox role is limited to reduction of the oxy-complex [5], giving rise to the nucleophilic peroxo-species, the reactive intermediate responsible for carbon-carbon bond scission [23,35]. An enhanced reduction rate of the oxy-complex is expected to increase the steady-state concentration of the peroxoanion, which in the absence of cyt b 5 is depleted through non-productive release of hydrogen peroxide.…”

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

“…3. This observation may contradict the conclusions about the active iron hydroperoxo species observed by resonance Raman spectroscopy (9,25). However, it is possible that the iron peroxohemiketal species reported in the resonance Raman study (9), i.e.…”

“…5), the signal-to-noise ratio of 18 O-incorporated acetate was three times greater than when (8,9). For clarity throughout the text, compound I is referred to interchangeably with FeO 3ϩ , and ferric peroxide is referred to interchangeably with FeO 2 Ϫ .…”

“…Although resonance Raman spectra of what is reported to be the human P450 17A1 FeO 2 Ϫ complex have recently been published (9,25), two caveats are as follows: (i) no cytochrome b 5 (for which the 17␣,20-lyase reaction is very dependent, e.g. Fig.…”