Active site proton delivery and the lyase activity of human CYP17A1

Abstract

Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20 carbon-carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17's physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17∝-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon-carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

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

“…Nanodiscs with CYP17A1 were assembled as documented [21]. Incorporation of CPR and cyt b 5 into purified CYP17A1 Nanodiscs was performed by direct addition of CPR and/or cyt b 5 in 2 to 4-fold molar excess, as documented [22–24]. …”

“…The resultant solution incubated at 37°C for 15 minutes. Substrate was added to each sample from methanolic stocks such that the final concentration was 420 µM [24]. Finally, ultrapure glycerol was added to a concentration of 15% (v/v); i.e., the final concentration of CYP17A1 was calculated to be 85 µM.…”

Membrane Anchor of Cytochrome P450 Reductase Suppresses the Uncoupling of Cytochrome P450

“…In Fig. 6, the generally accepted reaction cycle of CYP 39,46) is depicted. CPR has been suggested to supply electrons to substrate-bound and superoxide-bound ferric CYPs.…”

Structural insights into the function of steroidogenic cytochrome P450 17A1

“…Recent studies from two research groups have proposed that the peroxyanion species is the important species (Akhtar et al, 2011; Khatri et al, 2014), with the distal oxygen involved in a nucleophilic reaction and homolytic cleavage of the O-O bond resulting in deacetylation and generation of a free carbon radical. Subsequent hydrogen abstraction by the Fe(III)-O complex is proposed to afford the double bond yielding the ketone product.…”