Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

P450 CYP17A1 Variant with a Disordered Proton Shuttle Assembly Retains Peroxo-Mediated Lyase Efficiency.

Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C17 -C20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-Op -Ot fragment, facilitating peroxo- attack on the C20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.

Allosteric Interactions in Human Cytochrome P450 CYP3A4: The Role of Phenylalanine 213.

The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant. Using all-atom molecular dynamics simulations, we compared the dynamics of WT CYP3A4 and the F213A mutant incorporated into the lipid bilayer and the effect of the presence of the PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.

Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria.

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

Mechanism of drug-drug interactions mediated by human cytochrome P450 CYP3A4 monomer.

Using Nanodiscs, we quantitate the heterotropic interaction between two different drugs mediated by monomeric CYP3A4 incorporated into a nativelike membrane environment. The mechanism of this interaction is deciphered by global analysis of multiple-turnover experiments performed under identical conditions using the pure substrates progesterone (PGS) and carbamazepine (CBZ) and their mixtures. Activation of CBZ epoxidation and simultaneous inhibition of PGS hydroxylation are measured and quantitated through differences in their respective affinities for both a remote allosteric site and the productive catalytic site near the heme iron. Preferred binding of PGS at the allosteric site and a stronger preference for CBZ binding at the productive site give rise to a nontrivial drug-drug interaction. Molecular dynamics simulations indicate functionally important conformational changes caused by PGS binding at the allosteric site and by two CBZ molecules positioned inside the substrate binding pocket. Structural changes involving Phe-213, Phe-219, and Phe-241 are thought to be responsible for the observed synergetic effects and positive allosteric interactions between these two substrates. Such a mechanism is likely of general relevance to the mutual heterotropic effects caused by biologically active compounds that exhibit different patterns of interaction with the distinct allosteric and productive sites of CYP3A4, as well as other xenobiotic metabolizing cytochromes P450 that are also involved in drug-drug interactions. Importantly, this work demonstrates that a monomeric CYP3A4 can display the full spectrum of activation and cooperative effects that are observed in hepatic membranes.

Active site proton delivery and the lyase activity of human CYP17A1.

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.

Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers.

High-density lipoproteins (HDLs) play an important role in human health through the metabolism and trafficking of cholesterol as well as providing the feedstocks for steroid hormone biosynthesis. These particles contain proteins, primarily Apo-AI and phospholipid and progress through various structural forms including 'lipid-poor', 'discoidal' and 'spherical' entities as cholesterol esters and lipid are incorporated. The discoidal form of HDL is stabilized in solution by two encircling belts of Apo-AI. Previous protein engineering of the Apo-AI sequence has led to a series of amphipathic helical proteins, termed membrane scaffold proteins (MSPs), which have shown great value in assembling nanoscale soluble membrane bilayers, termed Nanodiscs, of homogeneous size and composition and in the assembly of numerous integral membrane proteins for biophysical and biochemical investigations. In this communication we document a protein engineering approach to generate and optimize an extended polypeptide MSP, which will self-assemble phospholipids into larger Nanodiscs with diameters of 16-17 nm. We extensively characterize these structures by size exclusion chromatography and solution X-ray scattering.

The critical iron-oxygen intermediate in human aromatase.

Aromatase (CYP19) is the target of several therapeutics used for breast cancer treatment and catalyzes the three-step conversion of androgens to estrogens, with an unusual C-C cleavage reaction in the third step. To better understand the CYP19 reaction, the oxy-ferrous complex of CYP19 with androstenedione substrate was cryotrapped, characterized by UV-vis spectroscopy, and cryoreduced to generate the next reaction cycle intermediate. EPR analysis revealed that the initial intermediate observed following cryoreduction is the unprotonated g(1)=2.254 peroxo-ferric intermediate, which is stable up to 180K. Upon gradual cryoannealing, the low-spin (g(1)=2.39) product complex is formed, with no evidence for accumulation of the g(1)=2.30 hydroperoxo-ferric intermediate. The relative stabilization of the peroxo-ferric heme and the lack of observed hydroperoxo-ferric heme distinguish CYP19 from other P450s, suggesting that the proton delivery pathway is more hindered in CYP19 than in most other P450s.