An epoxidation mechanism of carbamazepine by CYP3A4

Human CYP3A4 catalyzes the 10,11-epoxidation of carbamazepine (CBZ). However, the epoxide is less stable in terms of potential energy than hydroxides of the six-membered aromatic ring. To clarify the reason why CYP3A4 produces such an energetically unfavorable compound, the mechanism of epoxidation of CBZ by CYP3A4 was investigated by theoretical calculations. The reaction consisted of two elementary processes in which two C–O bonds were generated stepwise. The rate-determining step was the first one and the activation energy was 21.3 kcal/mol at the DFT (B3LYP/6-31G^**) level. The activation energy level of the first step of the 10,11-epoxidation was lower than that of the hydroxylation of the aromatic ring. For this reason, 10,11-epoxidation is more probable than hydroxylation of the aromatic ring, and only 10,11-epoxide is formed.     

Theoretical calculation of triazolam hydroxylation and endogenous steroid inhibition in the active site of CYP3A4

CYP3A4 has unusual kinetic characteristics because it has a large active site. CYP3A4 produced more 4-hydroxytriazolam than alpha-hydroxytriazolam at concentrations of more than 60 muM triazolam, and different steroids had different inhibitory effects on the system. To clarify these interesting observations, the interactions between substrate and substrate/steroid were investigated by theoretical calculations. When two triazolam molecules were docked into the active site, the distance between the O-atom and the 4-hydroxylated site was less than the distance to the alpha-hydroxylated site because of interaction between the two triazolam molecules. Estradiol inhibited both alpha- and 4-hydroxytriazolam formation by 50%. Dehydroepiandrosterone (DHEA) inhibited alpha-hydroxylation more than 4-hydroxytriazolam formation, whereas aldosterone had no effect. When one triazolam molecule and one steroid molecule were simultaneously docked, estradiol increased the distance between the O-atom and the two hydroxylated sites, DHEA only increased the distance between the O-atom and alpha-hydroxylated site, and aldosterone did not change the distances. The relevant angles of Fe-O-C in the hydroxylated site of triazolam also widened, together with increased distance. These findings indicate that formation of a substrate and substrate/effector complex in the active site may be a factor for determining the enzyme kinetic parameters of CYP3A4.     

A three-dimensional model of CYP19 aromatase for structure-based drug design

Aromatase (CYP450_arom, CYP19) is an enzyme responsible for converting the aliphatic androgens androstenedione and testosterone to the aromatic estrogens estrone and estradiol, respectively. These endogenous hormones are a key factor in cancer tumor formation and proliferation through a cascade starting from estrogen binding to estrogen receptor. To interfere with the overproduction of estrogens especially in tumor tissue, it is possible to inhibit aromatase activity. This can be achieved using aromatase inhibitors. In order to design novel aromatase inhibitors, it is necessary to have an understanding of the active site of aromatase. As no crystal structure of the enzyme has yet been published, we built a homology model of aromatase using the first crystallized mammalian cytochrome enzyme, rabbit 21-progesterone hydroxylase 2C5, as a template structure. The initial model was validated with exhaustive molecular dynamics simulation with and without the natural substrate androstenedione. The resulting enzyme–substrate complex shows very good stability and only two of the residues are in disallowed regions in a Ramachandran plot.     

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes: Gas chromatographic–mass spectrometric determination of metabolites

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17α-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17α-methyltestosterone, produced 6β-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography–mass spectrometry (GC–MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6β-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6β-hydroxyl metabolites with testosterone and 17α-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6β-hydroxylation.     

Metabolism of carbamazepine by CYP3A6: a model for in vitro drug interactions studies

Carbamazepine (CBZ) is widely used in the treatment of epilepsy. The drug is principally metabolized by CYPs to 10, 11-epoxy carbamazepine (CBZ-E) but this metabolite more toxic than the parent drug, does possess anticonvulsant properties. In humans, CYP3A4, CYP2C8 and CYP1A2 have been shown to be implicated in CBZ biotransformation. Our purpose was to establish an experimental model to determine the interaction of CBZ with other antiepileptic drugs. We first identified the CYP isoforms that metabolized CBZ in rabbit. We used liver microsomes from rabbit treated with various compounds known to induce principally some CYPs subfamilies. Having tested all the compounds we demonstrated that only the animals treated with CYP3A inducers were able to metabolize CBZ strongly. The CBZ biotransformation was inhibited by anti CYP3A antibodies. All the CYP3A subfamily substrates specifically decrease CBZ-E formation. In our experiment we did not observe any inhibition with CYP2C substrate. These data provide evidence that in rabbit the CYP3A subfamily is primarily involved in CBZ metabolism. Using this model we investigated the interaction of CBZ with phenobarbital, phenytoin, ethosuccimide, primidone, progabide, vigabatrin and lamotrigine.     

Differential effect of Cu2+ and Zn2+ on the formation and further metabolism of catechol estrogen by rat liver microsomes

The action of a number of different divalent metal ions on the rat liver microsomal release of 3H2O from estradiol and 2-hydroxyestradiol labeled with 3H at C-2 or C-4 was investigated. Cu2+ at low concentration (10 microM) produced a marked and specific inhibition of the 2-hydroxylation of estradiol with virtually no effect on the further oxidative activation of catechol estrogen. In contrast, Zn2+ inhibited the interaction of 2-hydroxyestradiol with microsomal protein as measured by the release of 3H from C-4 of the labeled steroids but did not influence 2-hydroxylation, except at high concentration. Other metal ions tested produced little or no change. Cu2+ inhibited the irreversible binding of estradiol to protein but activated this reaction with the catechol estrogen as substrate. The action of both Cu2+ and Zn2+ was reversed by glutathione. The differential effect of these metal ions on estrogen metabolism gives additional support for two different mechanisms in the cytochrome P-450-catalyzed formation of catechol estrogens and their further activation to form protein conjugates.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Investigation of ligand selectivity in CYP3A7 by molecular dynamics simulations

Cytochrome P450 (CYP) 3A7 plays a crucial role in the biotransformation of the metabolized endogenous and exogenous steroids. To compare the metabolic capabilities of CYP3A7–ligands complexes, three endogenous ligands were selected, namely dehydroepiandrosterone (DHEA), estrone, and estradiol. In this study, a three-dimensional model of CYP3A7 was constructed by homology modeling using the crystal structure of CYP3A4 as the template and refined by molecular dynamics simulation (MD). The docking method was adopted, combined with MD simulation and the molecular mechanics generalized born surface area method, to probe the ligand selectivity of CYP3A7. These results demonstrate that DHEA has the highest binding affinity, and the results of the binding free energy were in accordance with the experimental conclusion that estrone is better than estradiol. Moreover, several key residues responsible for substrate specificity were identified on the enzyme. Arg372 may be the most important residue due to the low interaction energies and the existence of hydrogen bond with DHEA throughout simulation. In addition, a cluster of Phe residues provides a hydrophobic environment to stabilize ligands. This study provides insights into the structural features of CYP3A7, which could contribute to further understanding of related protein structures and dynamics.     

CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding

Cytochrome p450c17 (CYP17) converts the C21 steroids pregnenolone and progesterone to the C19 androgen precursors dehydroepiandrosterone (DHEA) and androstenedione, respectively, via sequential 17alpha-hydroxylase and 17,20-lyase reactions. Disabling mutations in CYP17 cause combined 17alpha-hydroxylase/17,20-lyase deficiency, but rare missense mutations cause isolated loss of 17,20-lyase activity by disrupting interactions of redox partner proteins with CYP17. We studied an adolescent male with clinical and biochemical features of isolated 17,20-lyase deficiency, including micropenis, hypospadias, and gynecomastia, who is homozygous for CYP17 mutation E305G, which lies in the active site. When expressed in HEK-293 cells or Saccharomyces cerevisiae, mutation E305G retains 17alpha-hydroxylase activities, converting pregnenolone and progesterone to 17alpha-hydroxysteroids. However, mutation E305G lacks 17,20-lyase activity for the conversion of 17alpha-hydroxypregnenolone to DHEA, which is the dominant pathway to C19 steroids catalyzed by human CYP17 (the delta5-steroid pathway). In contrast, mutation E305G exhibits 11-fold greater catalytic efficiency (kcat/Km) for the cleavage of 17alpha-hydroxyprogesterone to androstenedione compared with wild-type CYP17. We conclude that mutation E305G selectively impairs 17,20-lyase activity for DHEA synthesis despite an increased capacity to form androstenedione. Mutation E305G provides genetic evidence that androstenedione formation from 17alpha-hydroxyprogesterone via the minor delta4-steroid pathway alone is not sufficient for complete formation of the male phenotype in humans.     

The use of transgenic cell lines for evaluating toxic metabolites of carbamazepine

Human lymphoblastoid cell lines transgenic for human CYP450s were evaluated for the identification of toxic metabolites of the anticonvulsant drug carbamazepine (CBZ). Human CYP450 isoforms expressed by these cell lines included 1A1, 1A2, 2E1, 2A6 and 3A4. A dose-dependent inhibition of population growth from 50–200 g/ml CBZ was detected by measuring cell number and respiration. The inhibition increased with the growth rate of the various lines, which correlated inversely with the presence of CYP450s, and may have been caused by CBZ itself. Cytotoxicity was observed only at the highest dose and in the line lacking transfected CYP450s. Microsomal preparations from hCYP3A4/OR cells converted CBZ into its principal oxidative metabolite, carbamazepine-10,11-epoxide (CBZ-E), at a rate of 630 pmol/min per mg protein, confirming a major role of CYP3A4 in this reaction. However, no CBZ-E (or any metabolite) was recovered from any whole-cell incubation even though hCYP3A4 cells readily converted testosterone to 6ß-hydroxytestosterone. This suggests that differences exist between whole-cell and microsomal preparations of lymphoblastoid cells in their ability to metabolize CBZ.Abbreviations BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide - CBZ carbamazepine - CBZ-E carbamazepine-10, 11-epoxide - CYP450 cytochrome P450 - CYP3A4 cytochrome P450, isoform 3A4 - DMSO dimethyl sulfoxide - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - MTT (3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl)tetrazolium - SIM selected-ion monitoring - TMS trimethylsilyl     

The Structure of Mycobacterium tuberculosis CYP125: MOLECULAR BASIS FOR CHOLESTEROL BINDING IN A P450 NEEDED FOR HOST INFECTION*

We report characterization and the crystal structure of the Mycobacterium tuberculosis cytochrome P450 CYP125, a P450 implicated in metabolism of host cholesterol and essential for establishing infection in mice. CYP125 is purified in a high spin form and undergoes both type I and II spectral shifts with various azole drugs. The 1.4-Å structure of ligand-free CYP125 reveals a “letterbox” active site cavity of dimensions appropriate for entry of a polycyclic sterol. A mixture of hexa-coordinate and penta-coordinate states could be discerned, with water binding as the 6th heme-ligand linked to conformation of the I-helix Val²⁶⁷ residue. Structures in complex with androstenedione and the antitubercular drug econazole reveal that binding of hydrophobic ligands occurs within the active site cavity. Due to the funnel shape of the active site near the heme, neither approaches the heme iron. A model of the cholesterol CYP125 complex shows that the alkyl side chain extends toward the heme iron, predicting hydroxylation of cholesterol C27. The alkyl chain is in close contact to Val²⁶⁷, suggesting a substrate binding-induced low- to high-spin transition coupled to reorientation of the latter residue. Reconstitution of CYP125 activity with a redox partner system revealed exclusively cholesterol 27-hydroxylation, consistent with structure and modeling. This activity may enable catabolism of host cholesterol or generation of immunomodulatory compounds that enable persistence in the host. This study reveals structural and catalytic properties of a potential M. tuberculosis drug target enzyme, and the likely mode by which the host-derived substrate is bound and hydroxylated.     

Genomic organization of a human 5beta-reductase and its pseudogene and substrate selectivity of the expressed enzyme

The enzyme 5beta-reductase catalyzes the reduction of the 4-ene of 3-ketosteroids, converting them into 5beta-dihydro-3-ketosteroids and, thus, could be involved in the metabolism of 4-cholestene-3-one, progesterone, 17~-hydroxyprogesterone, aldosterone, corticosterone, cortisol, 4-androstenedione, and testosterone. In this study, we report the genomic structure of a human 5beta-reductase gene, its tissue distribution, the characterization of an intronless pseudogene and the substrate selectivity of the enzyme. The gene coding for the active 5beta-reductase contains nine exons like most members of the aldo-keto reductase family, but the sequence covered by the gene, more than 42 kb, is much longer than the sequence of other members of this family. There are many large introns, especially introns 3, 4 and 7 that span approx. 7 kb, and intron 1 that contains more than 10 kb. Northern blot analysis showed three band sizes of 1.3, 2.2 and 2.7 kb. The 1.3 and 2.7 kb bands are highly expressed in the liver while weaker 2.2 and 1.3 kb bands have been observed in the testis and colon, respectively. We also identified an intronless gene having 86% homology with the 5beta-reductase cDNA sequence. Since its sequence contains many stop codons, this gene is most probably a pseudogene. To determine more precisely the substrate selectivity of the enzyme, we established a stable cell line expressing human 5beta-reductase in transformed embryonic kidney (HEK-293) cells. The transfected cells efficiently catalyze the transformation of progesterone, androstenedione, 17alpha-hydroxyprogesterone and testosterone. However, they catalyze much less efficiently the transformation of compounds containing an 11beta-hydroxy group, such as aldosterone, corticosterone and cortisol. In addition to its role in cholesterol catabolism, it is well recognized that 5beta-reductase inactivates active androgens. Indeed, 5beta-dihydrotestosterone (5beta-DHT), the product of the reduction of testosterone by 5beta-reductase, is not active while its 5~-isomer (DHT) is the most potent natural androgen. Recent findings show that 5beta-pregnanes are active ligands in the induction of CYP3A through the orphan receptor hPAR. Our results thus open an opportunity for studying the new role of 5beta-reductase in the formation of a new type of active steroids.     

Structural motif-based homology modeling of CYP27A1 and site-directed mutational analyses affecting vitamin D hydroxylation

Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D(3) and in the biosynthesis of bile acids. We have applied a systematic analysis of hydrogen bonding patterns in 11 prokaryotic and mammalian CYP crystal structures to construct a homology-based model of CYP27A1. Docking of vitamin D(3) structures into the active site of this model identified potential substrate contact residues in the F-helix, the beta-3 sheet, and the beta-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1alpha-hydroxyvitamin D(3) to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the beta-5 hairpin help define the shape of the substrate binding cavity and that this structure interacts with Phe-248 in the F-helix. Mutations directed toward the beta-3a strand suggested a possible heme-binding interaction centered on Asn-403 and a structural role for substrate contact residues Thr-402 and Ser-404.     

The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4

Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1'-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1'-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T(1) nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T(1) NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position 4 of the MDZ molecule was closer to the heme than the protons of the 1'-CH(3) group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1'-CH(3) position closer to the heme than position 4. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.     

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.     

The importance of residues in substrate recognition site 3 for the catalytic function of CYP2D25 (vitamin D 25-hydroxylase).

Porcine CYP2D25, microsomal vitamin D(3) 25-hydroxylase, catalyzes the essential first step in the bioactivation of the prohormone vitamin D(3). Although CYP2D25 shows a high degree of sequence identity with other members of the CYP2D subfamily, such as human CYP2D6, the vitamin D(3) 25-hydroxylase activity is a unique property among CYP2D enzymes. In addition to 25-hydroxylation, CYP2D25 also metabolizes the drug tolterodine. In this study, CYP2D25 was functionally expressed in the Saccharomyces cerevisiae W(R) strain and site-directed mutagenesis was used to study the role of substrate recognition site 3 (SRS-3) for the catalytic specificity of CYP2D25. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Western blot analysis of microsomes from transformed yeast cells showed that both the wild-type and mutant CYP2D25 were expressed at comparable levels. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained virtually unaffected. The results implicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D(3) metabolism.     

Scanning chimeragenesis: the approach used to change the substrate selectivity of fatty acid monooxygenase CYP102A1 to that of terpene ω-hydroxylase CYP4C7

CYP102A1 is a highly active, water-soluble, bacterial monooxygenase enzyme that contains both substrate-binding heme and diflavin reductase subunits, both in a single polypeptide. Recently we developed a procedure which uses the known structure of the substrate-bound heme domain of CYP102A1 and its sequence homology with a cytochrome P450 of unknown structure, both of which react with a common substrate but produce different products, to create recombinant enzymes which have substrate selectivity different from that of CYP102A1, and produce the product of the enzyme of unknown structure. Insect CYP4C7, a terpene hydroxylase from the cockroach, was chosen as the cytochrome P450 of unknown structure, and farnesol was chosen as the substrate. CYP102A1 oxidizes farnesol to three products (2,3-epoxyfarnesol, 10,11-epoxyfarnesol, and 9-hydroxyfarnesol), whereas CYP4C7 produces 12-hydroxyfarnesol as the major product. In earlier work it was found that the chimera C(78-82,F87L) showed a change in substrate selectivity from fatty acids to farnesol, and was approximately sixfold more active than wild-type CYP102A1 (Chen et al. in J Biol Inorg Chem 13:813–824, 2008), but neither it nor any other earlier chimera produced 12-hydroxyfarnesol. In this work we added amino acid residues 327–332, to create six new full-length, functional chimeric proteins. Four of these, the most active of which was C(78-82,F87L,328-330), produce 12-hydroxyfarnesol as the major product, with approximately twofold increase in turnover number as compared with wild-type CYP102A1 toward farnesol. Methylfarnesoate was metabolized to 12-hydroxymethylfarnesoate (70%) and 10,11-epoxymethylfarnesoate (juvenile hormone III) (30%). The latter is metabolized to 65% 12-hydroxy-10,11-epoxymethylfarnesoate and 35% 15-hydroxy-10,11-epoxymethylfarnesoate. Substitution of residues 328–330, APA, by VPL was crucial to accomplishing this change in product.     

Steroidal Metabolites Transformed by <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> Cultures Block Breast Cancer Estrogen Biosynthesis

Suspension of cultured cells of Marchantia polymorpha have the potential to hydrogenate the olefinic bonds present in androst-1,4-dien-3,17-dione (boldione, 1) to afford dihydroandrost-3,17-dione derivatives including: androst-4-ene-3,17-dione (androstenedione, 4-AD, 2), 5α-androstane-3,17-dione (androstenedione, AD, 4), and the less abundant metabolite 5α-androst-1-ene-3,17-dione (1-androstenedione, 1-AD, 3). After isolation and purification, these metabolites were characterized on the basis of spectroscopic analyses using 1D and 2D NMR as well as mass spectrometry. Cytotoxicity of the biotransformation products against breast adenocarcinoma cells (MCF-7) was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and cell death (apoptosis or necrosis) was assayed by acridine orange/ethidium bromide staining. Aromatase (cytochrome P450 19 enzyme, CYP19) inhibitory activity was measured by a tritiated water release assay and by direct measurement of bio-transformed steroids using the tritium labeled substrate ³H-androst-4-ene-3,17-dione. CYP19 mRNA expression in MCF-7 cells was analyzed by real-time PCR. Steroidal products 3 and 4 revealed a highly significant inhibition of MCF-7 cell growth that was predominantly due to apoptosis not necrosis. Steroidal products 3 and 4 are both potent inhibitors of aromatase activity and CYP19 mRNA expression, while 2 is a known substrate for aromatase. These data establish that metabolites 3 and 4 are potent chemical agents against breast cancer via aromatase inhibitory mechanism. Results were interpreted via virtual docking of the biotransformation products to the human placental aromatase active site.     

Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation.