Browse
Submit Data
Databases
API
Help

Dataset Information

33 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Tight binding of cytochrome b₅ to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis.

ABSTRACT: It has been recognized for >50 years that cytochrome b₅ (b₅) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b₅ is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b₅ function. One of the issues in studying b₅-P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b₅ that can be used in titrations. The labeled b₅ bound to WT P450 17A1 with a K_d of 2.5 nM and rapid kinetics, on the order of 1 s^-1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b₅ binding. K_d values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b₅ fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b₅:P450 17A1 complex resulted in a concentration-dependent partial restoration of b₅ fluorescence, indicative of a ternary P450:b₅:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b₅ complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

SUBMITTER: Kim D

PROVIDER: S-EPMC8080067 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Structural and kinetic basis of steroid 17?,20-lyase activity in teleost fish cytochrome P450 17A1 and its absence in cytochrome P450 17A2.

Project description:Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17?-hydroxylation and a subsequent 17?,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17?-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17?-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17?-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ?50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.

| S-EPMC4318999 | biostudies-literature

Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production.

Project description:Human cytochrome P450 17A1 is required for all androgen biosynthesis and is the target of abiraterone, a drug used widely to treat advanced prostate cancer. P450 17A1 catalyzes both 17-hydroxylation and subsequent 17,20-lyase reactions with pregnenolone, progesterone, and allopregnanolone. The presence of cytochrome b5 (b5) markedly stimulates the 17,20-lyase reaction, with little effect on 17-hydroxylation; however, the mechanism of this b5 effect is not known. We determined the influence of b5 on coupling efficiency-defined as the ratio of product formation to NADPH consumption-in a reconstituted system using these 3 pairs of substrates for the 2 reactions. Rates of NADPH consumption ranged from 4 to 13 nmol/min/nmol P450 with wild-type P450 17A1. For the 17-hydroxylase reaction, progesterone oxidation was the most tightly coupled (?50%) and negligibly changed upon addition of b5. Rates of NADPH consumption were similar for the 17-hydroxylase and corresponding 17,20-lyase reactions for each steroid series, and b5 only slightly increased NADPH consumption. For the 17,20-lyase reactions, b5 markedly increased product formation and coupling in parallel with all substrates, from 6% to 44% with the major substrate 17-hydroxypregnenolone. For the naturally occurring P450 17A1 mutations E305G and R347H, which impair 17,20-lyase activity, b5 failed to rescue the poor coupling with 17-hydroxypregnenolone (2-4%). When the conserved active-site threonine was mutated to alanine (T306A), both the activity and coupling were markedly decreased with all substrates. We conclude that b5 stimulation of the 17,20-lyase reaction primarily derives from more efficient use of NADPH for product formation rather than side products.

| S-EPMC5287367 | biostudies-literature

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1).

Project description:Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.

| S-EPMC9892303 | biostudies-literature

Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer.

Project description:The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase-selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel's direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor-bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC.

| S-EPMC5451248 | biostudies-literature

Steroidogenic cytochrome P450 17A1 structure and function.

Project description:Cytochrome P450 17A1 (CYP17A1) is a critical steroidogenic enzyme, essential for producing glucocorticoids and sex hormones. This review discusses the complex activity of CYP17A1, looking at its role in both the classical and backdoor steroidogenic pathways and the complex chemistry it carries out to perform both a hydroxylation reaction and a carbon-carbon cleavage, or lyase reaction. Functional and structural investigations have informed our knowledge of these two reactions. This review focuses on a few specific aspects of this discussion: the identities of reaction intermediates, the coordination of hydroxylation and lyase reactions, the effects of cytochrome b5, and conformational selection. These discussions improve understanding of CYP17A1 in a physiological setting, where CYP17A1 is implicated in a variety of steroidogenic diseases. This information can be used to improve ways in which CYP17A1 can be effectively modulated to treat diseases such as prostate and breast cancer, Cushing's syndrome, and glioblastoma.

| S-EPMC8087655 | biostudies-literature

Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes.

Project description:Cytochrome P450 (P450) 17A1 catalyzes the oxidations of progesterone and pregnenolone and is the major source of androgens. The enzyme catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction, and several mechanisms have been proposed for the latter step. Zebrafish P450 17A2 catalyzes only the 17α-hydroxylations. We previously reported high similarity of the crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1. Five residues near the heme, which differed, were changed. We also crystallized this five-residue zebrafish P450 17A1 mutant, and the active site still resembled the structure in the other proteins, with some important differences. These P450 17A1 and 17A2 mutants had catalytic profiles more similar to each other than did the wildtype proteins. Docking with these structures can explain several minor products, which require multiple enzyme conformations. The 17α-hydroperoxy (OOH) derivatives of the steroids were used as oxygen surrogates. Human P450 17A1 and zebrafish P450s 17A1 and P450 17A2 readily converted these to the lyase products in the absence of other proteins or cofactors (with catalytically competent kinetics) plus hydroxylated 17α-hydroxysteroids. The 17α-OOH results indicate that a "Compound I" (FeO3+) intermediate is capable of formation and can be used to rationalize the products. We conclude that zebrafish P450 17A2 is capable of lyase activity with the 17α-OOH steroids because it can achieve an appropriate conformation for lyase catalysis in this system that is precluded in the conventional reaction.

| S-EPMC5767860 | biostudies-literature

Stepwise binding of inhibitors to human cytochrome P450 17A1 and rapid kinetics of inhibition of androgen biosynthesis.

Project description:Cytochrome P450 (P450) 17A1 catalyzes the 17α-hydroxylation of progesterone and pregnenolone as well as the subsequent lyase cleavage of both products to generate androgens. However, the selective inhibition of the lyase reactions, particularly with 17α-hydroxy pregnenolone, remains a challenge for the treatment of prostate cancer. Here, we considered the mechanisms of inhibition of drugs that have been developed to inhibit P450 17A1, including ketoconazole, seviteronel, orteronel, and abiraterone, the only approved inhibitor used for prostate cancer therapy, as well as clotrimazole, known to inhibit P450 17A1. All five compounds bound to P450 17A1 in a multistep process, as observed spectrally, over a period of 10 to 30 s. However, no lags were observed for the onset of inhibition in rapid-quench experiments with any of these five compounds. Furthermore, the addition of substrate to inhibitor-P450 17A1 complexes led to an immediate formation of product, without a lag that could be attributed to conformational changes. Although abiraterone has been previously described as showing slow-onset inhibition (t1/2 = 30 min), we observed rapid and strong inhibition. These results are in contrast to inhibitors of P450 3A4, an enzyme with a larger active site in which complete inhibition is not observed with ketoconazole and clotrimazole until the changes are completed. Overall, our results indicate that both P450 17A1 reactions-17α-hydroxylation and lyase activity-are inhibited by the initial binding of any of these inhibitors, even though subsequent conformational changes occur.

| S-EPMC8350020 | biostudies-literature

The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1).

Project description:The steroid hydroxylation and carbon-carbon bond cleavage activities of cytochrome P450 17A1 (CYP17A1) are responsible for the production of glucocorticoids and androgens, respectively. The inhibition of androgen synthesis is an important strategy to treat androgen-dependent prostate cancer. We discuss the different enzymatic activities towards the various substrates of CYP17A1, demonstrating its promiscuity. Additionally, a novel interhelical interaction is proposed between the F-G loop and the B'-helix to explain the 16α-hydroxylase activity of human CYP17A1 with progesterone as the substrate. The techniques used by biochemists to study this important enzyme are also summarized. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.

| S-EPMC4456341 | biostudies-literature

Structures of human steroidogenic cytochrome P450 17A1 with substrates.

Project description:The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn(202) in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.

| S-EPMC4239641 | biostudies-literature

Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.

Project description:The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17?-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

| S-EPMC3675632 | biostudies-literature