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Epoxidation activities of human cytochromes P450c17 and P450c21.

Yoshimoto FK, Peng HM, Zhang H, Anderson SM, Auchus RJ - Biochemistry (2014)

Bottom Line: CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products.CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-ol-20-one) in 4:1 or 12:1 ratios, respectively.Catalase and superoxide dismutase did not prevent epoxide formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolism, Endocrinology, & Diabetes, Department of Internal Medicine and ‡Department of Pharmacology, University of Michigan , Ann Arbor, Michigan 48109, United States.

ABSTRACT
Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon-carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16α-hydroxylase activity of the enzymes.

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Spectrophotometric equilibrium binding titrationsof (A) CYP17A1or (B) CYP21A2 with progesterone and compound 1. Differencespectra were obtained upon titration of 200 nM purified P450 in 0.1M potassium phosphate buffer, pH 7.4, containing 20% glycerol (v/v)with substrates at 50–5000 nM. Plots of Δ(A392 – A420) for compound 1 and Δ(A386 – A420) for progesterone versus concentration ofsubstrate were fit to Michaelis–Menten equation using GraphPadPrism (see Experimental Procedures). Insetsshow individual spectra for progesterone (P4) and compound 1 at concentrations below plateau phase. The resulting Ks values were 540 nM and 60 nM for compound 1 and 740 nM and 100 nM for progesterone with CYP17A1 and CYP21A2,respectively.
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fig3: Spectrophotometric equilibrium binding titrationsof (A) CYP17A1or (B) CYP21A2 with progesterone and compound 1. Differencespectra were obtained upon titration of 200 nM purified P450 in 0.1M potassium phosphate buffer, pH 7.4, containing 20% glycerol (v/v)with substrates at 50–5000 nM. Plots of Δ(A392 – A420) for compound 1 and Δ(A386 – A420) for progesterone versus concentration ofsubstrate were fit to Michaelis–Menten equation using GraphPadPrism (see Experimental Procedures). Insetsshow individual spectra for progesterone (P4) and compound 1 at concentrations below plateau phase. The resulting Ks values were 540 nM and 60 nM for compound 1 and 740 nM and 100 nM for progesterone with CYP17A1 and CYP21A2,respectively.

Mentions: Titration of CYP17A1with compound 1 elicited a typical type I substrate-bindingspectrum similar to progesterone (Figure 3A).The presence of 16,17-double bond yielded a lower spectral changethan for progesterone, with ΔAmax about half that of progesterone (ε = 46 ± 2 mM–1 cm–1 for compound 1; ε = 94± 3 mM–1 cm–1 for progesterone),although with a higher affinity (Ks =540 nM for compound 1; Ks = 740 nM for progesterone). With CYP21A2, spectral titrations gavethe opposite pattern. Progesterone bound with high affinity (Ks = 60 nM) but gave a weak spectral change (ε= 38 ± 1 mM–1 cm–1), as waspreviously reported.24 With compound 1, the magnitude of the spectral change was about twice asgreat (ε = 77 ± 3 mM–1 cm–1), yet the affinity was poorer (Ks =100 nM, Figure 3B). Compound 1 is thus similar to progesterone in its affinity for CYP17A1 andCYP21A2 but somewhat different in its capacity to displace the axialwater molecule from the resting enzymes and to convert the heme ironsto the high-spin state.


Epoxidation activities of human cytochromes P450c17 and P450c21.

Yoshimoto FK, Peng HM, Zhang H, Anderson SM, Auchus RJ - Biochemistry (2014)

Spectrophotometric equilibrium binding titrationsof (A) CYP17A1or (B) CYP21A2 with progesterone and compound 1. Differencespectra were obtained upon titration of 200 nM purified P450 in 0.1M potassium phosphate buffer, pH 7.4, containing 20% glycerol (v/v)with substrates at 50–5000 nM. Plots of Δ(A392 – A420) for compound 1 and Δ(A386 – A420) for progesterone versus concentration ofsubstrate were fit to Michaelis–Menten equation using GraphPadPrism (see Experimental Procedures). Insetsshow individual spectra for progesterone (P4) and compound 1 at concentrations below plateau phase. The resulting Ks values were 540 nM and 60 nM for compound 1 and 740 nM and 100 nM for progesterone with CYP17A1 and CYP21A2,respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263428&req=5

fig3: Spectrophotometric equilibrium binding titrationsof (A) CYP17A1or (B) CYP21A2 with progesterone and compound 1. Differencespectra were obtained upon titration of 200 nM purified P450 in 0.1M potassium phosphate buffer, pH 7.4, containing 20% glycerol (v/v)with substrates at 50–5000 nM. Plots of Δ(A392 – A420) for compound 1 and Δ(A386 – A420) for progesterone versus concentration ofsubstrate were fit to Michaelis–Menten equation using GraphPadPrism (see Experimental Procedures). Insetsshow individual spectra for progesterone (P4) and compound 1 at concentrations below plateau phase. The resulting Ks values were 540 nM and 60 nM for compound 1 and 740 nM and 100 nM for progesterone with CYP17A1 and CYP21A2,respectively.
Mentions: Titration of CYP17A1with compound 1 elicited a typical type I substrate-bindingspectrum similar to progesterone (Figure 3A).The presence of 16,17-double bond yielded a lower spectral changethan for progesterone, with ΔAmax about half that of progesterone (ε = 46 ± 2 mM–1 cm–1 for compound 1; ε = 94± 3 mM–1 cm–1 for progesterone),although with a higher affinity (Ks =540 nM for compound 1; Ks = 740 nM for progesterone). With CYP21A2, spectral titrations gavethe opposite pattern. Progesterone bound with high affinity (Ks = 60 nM) but gave a weak spectral change (ε= 38 ± 1 mM–1 cm–1), as waspreviously reported.24 With compound 1, the magnitude of the spectral change was about twice asgreat (ε = 77 ± 3 mM–1 cm–1), yet the affinity was poorer (Ks =100 nM, Figure 3B). Compound 1 is thus similar to progesterone in its affinity for CYP17A1 andCYP21A2 but somewhat different in its capacity to displace the axialwater molecule from the resting enzymes and to convert the heme ironsto the high-spin state.

Bottom Line: CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products.CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-ol-20-one) in 4:1 or 12:1 ratios, respectively.Catalase and superoxide dismutase did not prevent epoxide formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolism, Endocrinology, & Diabetes, Department of Internal Medicine and ‡Department of Pharmacology, University of Michigan , Ann Arbor, Michigan 48109, United States.

ABSTRACT
Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon-carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16α-hydroxylase activity of the enzymes.

Show MeSH
Related in: MedlinePlus