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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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Chem3D Software was used to minimize the energyof the 16-dehydroprogesterone(compound 1) and progesterone (MM2) structures. Progesteroneis shown in red, and compound 1 is shown in blue. The“Fast Overlay” method was used to overlay the two structures.After the “Fast Overlay” technique, the optimal distancemeasurement for the C13, C14, and C18 carbon atoms on both progesteroneand 16-dehydroprogesterone structures was set to 0 Å, and thestructures were manually minimized under the overlay option. Thismanual overlay based on atoms C13, C14, and C18 indicates that theintroduction of the double bond between C16 and C17 positions theC21-carbon atom slightly closer to the original 17-position of progesterone.The distances between the progesterone 17-hydrogen atom and the C21-carbonatom of progesterone and of compound 1 are 3.032 and2.872 Å, respectively. The distances between the progesterone17-hydrogen atom and the C16-carbon atom of progesterone and of compound 1 are 2.185 and 2.327 Å, respectively.
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fig4: Chem3D Software was used to minimize the energyof the 16-dehydroprogesterone(compound 1) and progesterone (MM2) structures. Progesteroneis shown in red, and compound 1 is shown in blue. The“Fast Overlay” method was used to overlay the two structures.After the “Fast Overlay” technique, the optimal distancemeasurement for the C13, C14, and C18 carbon atoms on both progesteroneand 16-dehydroprogesterone structures was set to 0 Å, and thestructures were manually minimized under the overlay option. Thismanual overlay based on atoms C13, C14, and C18 indicates that theintroduction of the double bond between C16 and C17 positions theC21-carbon atom slightly closer to the original 17-position of progesterone.The distances between the progesterone 17-hydrogen atom and the C21-carbonatom of progesterone and of compound 1 are 3.032 and2.872 Å, respectively. The distances between the progesterone17-hydrogen atom and the C16-carbon atom of progesterone and of compound 1 are 2.185 and 2.327 Å, respectively.

Mentions: The alternative site of reactivity for CYP17A1 in the vicinityof the double bond between C-16 and C-17 in compound 1 is C-21. All enzymes studied readily 21-hydroxylated compound 1, even those with low progesterone 21-hydroxylase activity(Tables 1 and 2). Itis likely that the reduced 16α,17-epoxidase activity of CYP17A1mutation A105L also reflects the enhanced progesterone 21-hydroxylaseactivity of this enzyme, leading to greater metabolic switching toC-21. These data are consistent with a model in which substrate-bindingtrajectories for CYP17A1 render the hydrogen atoms at C-16, C-17,and C-21 all accessible for abstraction, but the regiochemical activitiestoward any given substrate reflect the reactivity of these positions,as well as the residence times within a critical distance from theheme oxygen. We previously suggested that the regiochemistry of CYP17A1primarily reflects the stability of the carbon-based radicals generatedafter hydrogen atom abstraction in this part of the molecule: C-17> C-16 > C-21, as carbon radical stability follows 3° >2°> 1°.5 With this model, reactivityat C-21 is not sterically precluded but rather suppressed by the greaterreactivity at C-16 and C-17. Metabolic switching to C-21 emerged asthe primary reaction pathway when the substrate was engineered toprevent 16α- and 17-hydroxylation, and all enzymes also epoxidizedthe double bond at C-16. Our data do not allow us to determine therelative contributions of changes in substrate trajectories and ofdifferences in the activation barrier for the epoxidation pathwayversus the predominant 21-hydroxylation pathway. The similar but moredisparate product distributions with compound 5 favorthe conclusion that substrate modifications are primarily uncoveringoccult reactivities rather than markedly altering substrate-bindingtrajectories. Energy minimization with Chem3D Software demonstratesthat the hydrogen atoms of C-21 in compound 1 are movedslightly (0.16 Å) closer to C-17 compared to progesterone (Figure 4), but this change is small compared to the markedincrease in 21-hydroxylation.


Epoxidation activities of human cytochromes P450c17 and P450c21.

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

Chem3D Software was used to minimize the energyof the 16-dehydroprogesterone(compound 1) and progesterone (MM2) structures. Progesteroneis shown in red, and compound 1 is shown in blue. The“Fast Overlay” method was used to overlay the two structures.After the “Fast Overlay” technique, the optimal distancemeasurement for the C13, C14, and C18 carbon atoms on both progesteroneand 16-dehydroprogesterone structures was set to 0 Å, and thestructures were manually minimized under the overlay option. Thismanual overlay based on atoms C13, C14, and C18 indicates that theintroduction of the double bond between C16 and C17 positions theC21-carbon atom slightly closer to the original 17-position of progesterone.The distances between the progesterone 17-hydrogen atom and the C21-carbonatom of progesterone and of compound 1 are 3.032 and2.872 Å, respectively. The distances between the progesterone17-hydrogen atom and the C16-carbon atom of progesterone and of compound 1 are 2.185 and 2.327 Å, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263428&req=5

fig4: Chem3D Software was used to minimize the energyof the 16-dehydroprogesterone(compound 1) and progesterone (MM2) structures. Progesteroneis shown in red, and compound 1 is shown in blue. The“Fast Overlay” method was used to overlay the two structures.After the “Fast Overlay” technique, the optimal distancemeasurement for the C13, C14, and C18 carbon atoms on both progesteroneand 16-dehydroprogesterone structures was set to 0 Å, and thestructures were manually minimized under the overlay option. Thismanual overlay based on atoms C13, C14, and C18 indicates that theintroduction of the double bond between C16 and C17 positions theC21-carbon atom slightly closer to the original 17-position of progesterone.The distances between the progesterone 17-hydrogen atom and the C21-carbonatom of progesterone and of compound 1 are 3.032 and2.872 Å, respectively. The distances between the progesterone17-hydrogen atom and the C16-carbon atom of progesterone and of compound 1 are 2.185 and 2.327 Å, respectively.
Mentions: The alternative site of reactivity for CYP17A1 in the vicinityof the double bond between C-16 and C-17 in compound 1 is C-21. All enzymes studied readily 21-hydroxylated compound 1, even those with low progesterone 21-hydroxylase activity(Tables 1 and 2). Itis likely that the reduced 16α,17-epoxidase activity of CYP17A1mutation A105L also reflects the enhanced progesterone 21-hydroxylaseactivity of this enzyme, leading to greater metabolic switching toC-21. These data are consistent with a model in which substrate-bindingtrajectories for CYP17A1 render the hydrogen atoms at C-16, C-17,and C-21 all accessible for abstraction, but the regiochemical activitiestoward any given substrate reflect the reactivity of these positions,as well as the residence times within a critical distance from theheme oxygen. We previously suggested that the regiochemistry of CYP17A1primarily reflects the stability of the carbon-based radicals generatedafter hydrogen atom abstraction in this part of the molecule: C-17> C-16 > C-21, as carbon radical stability follows 3° >2°> 1°.5 With this model, reactivityat C-21 is not sterically precluded but rather suppressed by the greaterreactivity at C-16 and C-17. Metabolic switching to C-21 emerged asthe primary reaction pathway when the substrate was engineered toprevent 16α- and 17-hydroxylation, and all enzymes also epoxidizedthe double bond at C-16. Our data do not allow us to determine therelative contributions of changes in substrate trajectories and ofdifferences in the activation barrier for the epoxidation pathwayversus the predominant 21-hydroxylation pathway. The similar but moredisparate product distributions with compound 5 favorthe conclusion that substrate modifications are primarily uncoveringoccult reactivities rather than markedly altering substrate-bindingtrajectories. Energy minimization with Chem3D Software demonstratesthat the hydrogen atoms of C-21 in compound 1 are movedslightly (0.16 Å) closer to C-17 compared to progesterone (Figure 4), but this change is small compared to the markedincrease in 21-hydroxylation.

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