Human Cytochrome P450 17A1 Conformational Selection

Estrada, D. Fernando; Skinner, Andria L.; Laurence, Jennifer S.; Scott, Emily E.

doi:10.1074/jbc.m114.560144

Cited by 67 publications

(91 citation statements)

References 44 publications

(53 reference statements)

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“…For all three isoforms, step-wise addition of CYP24A1-clotrimazole induced pronounced broadening of the NMR line shape for all 15 N-Adx resonances (Figure 2A), with approximately 30% of the original peak intensities remaining upon addition of 0.2-0.35 molar equivalents of P450 ( Figure 2B). Similar broadening, consistent with a protein interaction in the intermediate chemical exchange NMR time regime, has been observed in other P450 accessory protein complexes (21,22,25,26).While these titrations reveal a pattern of predominantly uniform broadening for Adx backbone resonances when the P450 is present, the ratios measured at 0.35 molar equivalents of human CYP24A1, 0.25 molar equivalents of rat CYP24A1, and 0.20 molar equivalents of opossum CYP24A1, generally presented a similar pattern of peak broadening ( Figure 2B). Modest differences in the extent of broadening were observed for residues in helix-3 (Asp-72 to Asp-79) as determined by ratios that deviated more than one standard deviation from the mean ( Figure 2B, highlighted in red), indicating a change in the environment in this region relative to the rest of Adx.…”

supporting

confidence: 63%

“…In a previous study, the high affinity inhibitor abiraterone was used to stabilize the catalytic domain of the steroidogonic enzyme CYP17A1 (21,22), thus conferring sufficient sample solubility as required for the acquisition of two dimensional and three dimensional NMR data. A similar approach was utilized here, in which incorporation of the P450 inhibitor clotrimazole resulted in enhanced bacterial expression of recombinant CYP24A1 isoforms when supplemented in growth media as well as conferring enhanced stability and solubility in NMR samples.…”

Section: Resultsmentioning

confidence: 99%

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The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

Estrada

2018

Journal of Biological Chemistry

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Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH) 2 D3. Its function relies on formation of a P450-redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx-CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450-Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1-Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH) 2 D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge-neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.Mitochondrial cytochromes P450 (P450) are responsible for a host of biological reactions, including steps necessary in steroid and vitamin D metabolism. Similar to microsomal P450s, their function requires the transfer of two electrons, delivered sequentially, in order to generate the active oxidizing species necessary for completion of one cycle of P450 catalysis (1). However, in contrast to microsomal P450 enzymes, for which reduction of P450 can be achieved by binding either a P450 oxidoreductase or, in some cases, the small heme protein cytochrome b 5 , mitochondrial enzymes rely entirely on formation of a transient complex with the soluble ferredoxin protein, Adrenodoxin (Adx). Adx folds into a compact structure consisting of three short α-helices and five anti-parallel β strands, with a [2Fe-2S] cluster coordinated near solvent accessible surfaces between helix-1 and helix-3 (2,3). Changes in the C-terminal region of bovine Adx have been shown to participate in mediating a monomer to dimer transition in response to reduction of the [2Fe-2S] cluster (2), thereby suggesting that dimers of Adx are functionally relevant.While the prevailing evidence suggests that complex formation between Adx with P450 relies on salt bridge interactions between the side chains of acidic residues on helix-3 (Asp-72 - Cytoch...

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supporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

Estrada

2018

Journal of Biological Chemistry

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“…Perhaps consistent with this view is the more efficient pregnenolone 17␣-hydroxylation reaction with P450 17A2 (Table 1), which might be the result of an enhanced conversion of the ferric peroxide (Fe (II) O 2 Ϫ ) to compound I in the catalytic cycle. However, we were unable to (59). In considering fish P450 17A1 and 17A2, it is possible that P450 17A1 adopts additional conformations (compared with P450 17A2), allowing it to catalyze the 17␣,20-lyase reaction in addition to 17␣-hydroxylation.…”

Section: Discussionmentioning

confidence: 97%

“…We propose that this amino acid residue of P450 17A1 is necessary for the lyase activity (although others may also cause this) and that multiple substitutions of P450 17A2 are probably needed to confer lyase activity to this protein. Human P450 17A1 Arg-358 has been implicated in b 5 binding through NMR, chemical cross-linking, and other techniques (59,72,73). However, zebrafish P450 17A1 shows only ϳ2-fold stimulation by b 5 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

Pallan

Nagy

Lei

et al. 2015

Journal of Biological Chemistry

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Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase reactions. A second fish P450, 17A2 (51% identical), catalyzes only 17␣-hydroxylation. Results: Crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1 are very similar. Conclusion: In kinetic analysis, the two-step oxidation of progesterone is more distributive than for pregnenolone. Significance: Small structural differences are associated with activities of the two fish P450s.

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“…This observation could explain the substrate selectivity of the lyase reaction and the increased 17,20-lyase activity after the allosteric binding of cytochrome b5. NMR studies have already established that b5 binds differently to CYP17A1 depending on whether the substrate is pregnenolone or 17α-hydroxypregnenolone [34] . Cytochrome b5 could alter the positioning of 17α-hydroxypregnenolone to the second position, thus increasing the rate of the lyase reaction.…”

Section: Cyp17a1mentioning

confidence: 99%

Androgen-AR axis in primary and metastatic prostate cancer: chasing steroidogenic enzymes for therapeutic intervention

Pippione¹,

Boschi²,

Pors³

et al. 2017

JCMT

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Androgens play an important role in prostate cancer (PCa) development and progression. Although androgen deprivation therapy remains the front-line treatment for advanced prostate cancer, patients eventually relapse with the lethal form of the disease. The prostate tumor microenvironment is characterised by elevated tissue androgens that are capable of activating the androgen receptor (AR). Inhibiting the steroidogenic enzymes that play vital roles in the biosynthesis of testosterone (T) and dihydrotestosterone (DHT) seems to be an attractive strategy for PCa therapies. Emerging data suggest a role for the enzymes mediating pre-receptor control of T and DHT biosynthesis by alternative pathways in controlling intratumoral androgen levels, and thereby influencing PCa progression. This supports the idea for the development of multi-targeting strategies, involving both dual and multiple inhibitors of androgen-metabolising enzymes that are able to affect androgen synthesis and signalling at different points in the biosynthesis. In this review, we will focus on CYP17A1, AKR1C3, HSD17B3 and SRD5A, as these enzymes play essential roles in all the three androgenic pathways. We will review also the AR as an additional target for the design of bifunctional drugs. Targeting intracrine androgens and AKR1C3 have potential to overcome enzalutamide and abiraterone resistance and improve survival of advanced prostate cancer patients.

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Human Cytochrome P450 17A1 Conformational Selection

Cited by 67 publications

References 44 publications

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

Androgen-AR axis in primary and metastatic prostate cancer: chasing steroidogenic enzymes for therapeutic intervention

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