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, 443 (1), 179-84

Active Site Proton Delivery and the Lyase Activity of Human CYP17A1


Active Site Proton Delivery and the Lyase Activity of Human CYP17A1

Yogan Khatri et al. Biochem Biophys Res Commun.


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.

Keywords: 17α-hydroxy-pregnenolone; 17α-hydroxy-progesterone; AD; CYP17A1; Compound I; Cpd I; DHEA; Nucelophilic attack; OH-PREG; OH-PROG; PREG; PROG; Proton delivery; T306A; androstenedione; cyt-b(5); cytochrome b(5); dehydroepiandrosterone; pregnenolone; progesterone.


Figure 1
Figure 1. A reaction cycle of cytochrome P450 catalysis
The seven steps indicating the binding of substrate (1), formation of a high spin ferric state (2), transfer of an electron from the redox partner to form the ferrous protein (3), generation of the ferrous oxygenated (oxy-ferrous) state (4), reduction to form the peroxo states after receiving second electron (5a), transfer of proton to the distal oxygen atom and production of the “hydroperoxo” state (5b), a second protonation and formation of a higher valent metal-oxo species “Compound I” (Cpd I) (6), and the hydroxylation and release of product (7) of P450 catalysis are illustrated. The three unproductive pathways are shown in grey.
Figure 2
Figure 2. Comparative study on 17-α-hydroxylase activity of pregnenolone and progesterone by wild type CYP17A1 and T306A
Determination of rate of NADPH oxidation (A) and the rate of conversion of pregnenolone (Δ5-steroid) and progesterone (Δ4-steroid) to the corresponding 17-OH-product (B) are shown. The black and open bars represent the WT and T306A, respectively. The coupling efficiency (C) of WT and T306A during the hydroxylation of Δ5- (stripped-bar) and Δ4-steroids (grey bar) are illustrated. The number on the bar represents the values of the activity.
Figure 3
Figure 3. Comparison of NADPH oxidation and coupling efficiency during 17,20-lyase activity by wild type CYP17A1 and T306A
Determination of rate of NADPH oxidation (black bar) and percentage of coupling (white bar) for 17-OH-PREG and 17-OH-PROG for wild type CYPC17A1 and T306A are shown. The number on the bar represents the corresponding value related with the unit on the y-axis. The error bar represents the standard deviation of 6–8 independent measurements.
Figure 4
Figure 4. Cytochrome b5 effect on CYP17A1 lyase activity
The rate conversion of 17-OH-PREG to DHEA and 17-OH-PROG to AD was measured in the presence (black bar) and absence (white bar) of cytochrome b5. The number on the bar represents the rate of conversion of the substrate and the error bar the standard deviation of 6–8 independent measurements.

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