Characterization of three cytochrome P450s purified from renal microsomes of untreated male rats and comparison with human renal cytochrome P450

Three renal cytochrome P450s (P450 K-2, K-4, and K-5) were purified from renal microsomes of untreated male rats. Also, the human renal cytochrome P450 (P450 HK) was partially purified from renal microsomes and its properties were compared with those of the rat renal cytochrome P450s. The molecular weight of P450 K-2, K-4, and K-5 was 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of P450 K-2, K-4, and K-5 were at 449, 451, and 452 nm, respectively. NH2-terminal sequence analysis of P450 K-2, K-4, and K-5 showed that these forms were different from hepatic cytochrome P450s purified previously. P450 K-2, K-4, and K-5 catalyzed the O-dealkylation of 7-ethoxycoumarin but were not efficient in the hydroxylation of testosterone. Aminopyrine was metabolized by P450 K-2 and K-4 but not by P450 K-5. Lauric acid was metabolized efficiently by all of these forms in the presence of cytochrome b5. The regiospecificity of these forms toward lauric acid was different. P450 K-2 hydroxylated lauric acid only at the (omega-1)-position, not at the omega-position. P450 K-4 and K-5 hydroxylated lauric acid at both the omega- and (omega-1)-positions. The ratios of omega/(omega-1)-hydroxylation activity of P450 K-4 and K-5 were 2.5 and 7.8, respectively. Human P450 HK was purified 220-fold and its specific content was 2.0 nmol/mg of protein. The Soret maxima of P450 HK were at 418 nm for the oxidized form, 416 nm for the reduced form, and 450 nm for the CO-reduced form. P450 HK catalyzed the O-dealkylation of 7-ethoxycoumarin but was not efficient in aminopyrine N-demethylation or testosterone hydroxylation. P450 HK had high lauric acid omega- and (omega-1)-hydroxylation activities in the presence of cytochrome b5, especially omega-hydroxylation. These properties resembled those of P450 K-5 most closely. Anti-P450 K-5 antibody cross-reacted with P450 HK as well as P450 K-5 and only one band was stained on immunostained Western blotting for partially purified P450 HK. The molecular weight of P450 HK was 52,000 on Western blotting.     

Rearrangement reactions catalyzed by cytochrome P450s

Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, “NIH” and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants.     

Phosphorylation of cytochrome P450: regulation by cytochrome b5

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.     

Quantitative evaluation of bromodichloromethane metabolism by recombinant rat and human cytochrome P450s

We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. Earlier work identified CYP2E1, CYP2B1/2 and CYP1A2 as activating enzymes necessary for hepatotoxicity in rat. In order to extend an existing PBPK model for rat to include a capability for extrapolation to humans, it is necessary to evaluate quantitatively the principal metabolic pathways in both species. We have conducted in vitro experiments using recombinant preparations of the three rat CYP isoenzymes mentioned above and for CYP2C11 and CYP3A1 as well. Similar experiments have been performed with human recombinant isoenzymes for CYP2E1, CYP1A2, CYP2A6, CYP2B6, CYP2D6 and CYP3A4. Results indicate that the principal metabolizing enzymes in rat are those identified previously, CYP2E1, CYP2B1/2 and CYP1A2. CYP3A1 may also have some activity. In human, CYP2E1, CYP1A2 and CYP3A4 show substantial activity, and CYP2A6 also measurably metabolizes BDCM. In both species, CYP2E1 is the low K(m) isoenzyme, with K(m) approximately 27-fold lower than those for the isoenzymes with the next lowest K(m). In addition, the metabolic parameters, K(m) and k(cat), for rat and human CYP2E1 were nearly identical. The metabolic parameters for CYP1A2, the only other isoenzyme active in both species, were not similar across species. In addition, calculations based on the kinetic constants obtained are compared to results from two in vivo experiments to show that the in vitro kinetic data is relevant to in vivo exposures. We conclude that although several CYPs metabolize BDCM, at low concentration/exposure, BDCM metabolism is dominated by CYP2E1 in both rat and human, but that other isoenzymes can be important at higher concentrations. We further conclude that the kinetic data are consistent with existing in vivo results.     

Alpha-tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic alpha-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic alpha-tocopherol metabolism and excretion, rats received daily subcutaneous alpha-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver alpha-tocopherol concentrations increased from 12 +/- 1 nmol/g (mean +/- SE) to 819 +/- 74 (Day 3), decreased at Day 9 (486 +/- 67), and continued to decrease through Day 18 (338 +/- 37). alpha-Tocopherol metabolites and their intermediates increased and decreased similarly to alpha-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic alpha-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver alpha-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic alpha-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.     

Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast

Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.     

A world of cytochrome P450s

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.     

Region-specific regulation of cytochrome P450 aromatase messenger ribonucleic acid by androgen in brains of male rhesus monkeys

We demonstrated previously that testosterone regulates aromatase activity in the anterior/dorsolateral hypothalamus of male rhesus macaques. To determine the level of the androgen effect, we developed a ribonuclease protection assay to study the effects of testosterone or dihydrotestosterone (DHT) on aromatase (P450(AROM)) mRNA in selected brain areas. Adult male rhesus monkeys were treated with testosterone or DHT. Steroids in serum were quantified by RIA. Fourteen brain regions were analyzed for P450(AROM) mRNA. Significant elevations of its message over controls (P<0.05) were found in the medial preoptic area/anterior hypothalamus of both androgen treatment groups and the medial basal hypothalamus of the testosterone-treated males. Other brain areas were not affected by androgen treatment. We conclude that testosterone and DHT regulate P450(AROM) mRNA in brain regions that mediate reproductive behaviors and gonadotropin release. The P450(AROM) mRNA of other brain areas is not androgen dependent. Brain-derived estrogens may also be important for maintaining neural circuitry in brain areas not related to reproduction. The control of P450(AROM) mRNA in these areas may differ from what we report here, but it is equally important to understand the function of in situ estrogen formation in these areas.     

Testosterone-mediated regulation of mouse renal cytochrome P-450 isoenzymes.

We have studied the extent to which mouse renal cytochrome P-450 isoenzymes are sexually differentiated, and the factor(s) regulating this dimorphism. Intriguingly, sex differences were not seen in the expression of a single cytochrome P-450 enzyme, but were observed in the expression of all P-450 isoenzymes detectable, encoded by six gene families or sub-families. This effect was mediated by testosterone, which had the capacity to both induce and repress P-450 gene expression, and which was independent of growth hormone. The changes in protein content were mirrored in all but one case by changes in the levels of mRNA, indicating that these genes contain hormone-responsive elements. These findings are consistent with numerous reports of sex differences in the susceptibility of the mouse kidney to the toxic and carcinogenic effects of drugs and environmental chemicals, many of which are metabolized to cytotoxic products by the cytochrome P-450-dependent mono-oxygenases. These data imply that circulating androgen levels will be an important factor in susceptibility of the kidney to toxic or carcinogenic compounds which require metabolic activation.     

Metabolons involving plant cytochrome P450s

Arranging biological processes into “compartments” is a key feature of all eukaryotic cells. Through this mechanism, cells can drastically increase metabolic efficiency and manage complex cellular processes more efficiently, saving space and energy. Compartmentation at the molecular level is mediated by metabolons. A metabolon is an ordered protein complex of sequential metabolic enzymes and associated cellular structural elements. The sub-cellular organization of enzymes involved in the synthesis and storage of plant natural products appears to involve the anchoring of metabolons by cytochrome P450 monooxygenases (P450s) to specific domains of the endoplasmic reticulum (ER) membrane. This review focuses on the current evidence supporting the organization of metabolons around P450s on the surface of the ER. We␣outline direct and indirect experimental data that describes P450 enzymes in the phenylpropanoid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. We also discuss the limitations and future directions of metabolon research and the potential for application to metabolic engineering endeavors.     

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homology modeling of plant cytochrome P450s

Plant P450 monooxygenases represent the largest family of plant proteins and the largest collection of P450s available for comparative studies and biotechnological applications. They have been shown to catalyze a diverse array of difficult chemical reactions and have demonstrated potential to be used in pharmacological, agronomic and phytoremediative applications. Central to our use of these catalytically competent enzymes is the need to understand their interactions with substrates. Because most characterized plant P450s are membrane-bound proteins that are resistant to standard X-ray and NMR structure determinations, homology modeling represents a reliable and relatively rapid alternative method for analyzing structure-function relationships and predicting substrates for many P450s that are only now being characterized. These methods, which are being widely used in mammalian P450 structure-function studies, can allow plant biologists to define critical residues interacting with substrates and, in a directed fashion, alter the reactivities of individual monooxygenases. The homology modelings that have been done on a limited number of plant P450s and the site-directed mutations that validate them indicate that current modeling and substrate docking procedures are capable of providing structural explanations for sequence variants as well as for predicting functional characteristics of undefined P450s.     

Hormonal regulation of androgen production by the Leydig cell

The control of androgen production by the Leydig cell is dependent upon the episodic secretion of hormone (LH), which is released from the anterior pituitary gland in pulses of high biological activity. This mode of episodic LH secretion supports steroidogenic enzyme activity in the testis through interaction with LH receptors and stimulation of the adenylate cyclase/protein kinase sequence, leading to phosphorylation of key intermediates in the steroid biosynthetic pathway. The plasma membrane events that are rapidly activated by the specific interaction of LH or hCG with Leydig cell receptors include increased binding of guanyl nucleotide, and stimulation of cAMP-independent, Ca²⁺dependent phosphorylation of a 44,500 Mr protein, with the characteristics of the adenylate cyclase nucleotide regulatory unit. Hormonal activation of adenylate cyclase is affected by Ca²⁺ with the same concentration-dependence, suggesting that nucleotide-induced phosphorylation is related to activation of the catalytic cyclase unit.In addition to the characteristic increases in pregnenolone synthesis and androgen production, gonadotropin-stimulated Leydig cells show prominent changes in LH receptor content and steroidogenic activity that modify their subsequent responses to hormonal signals. Thus, after exposure to increased LH and hCG levels in vivo and in vitro, LH receptors show an initial transient increase (up-regulation) followed by a marked decrease (down-regulation) and a prolonged depletion of LH receptor sites. Large doses of hCG cause “early” (prior to pregnenolone) and “late” steroidogenic lesions (17α-hydroxylase, 17–20 desmolase) that are independent of receptor loss. The early lesion is partly due to reduced activity of HMG CoA reductase, and is mainly attributable to the increased activity of an inhibitory protein factor that modulates the activity of cholesterol side chain cleavage enzyme in Leydig cell mitochondria. In contrast, the late steroidogenic lesion is related to the nuclear actions of E₂ produced during hormonal action. After hCG stimulation, an increase in nuclear E₂ binding was accompanied by an early rise of RNA polymerase activities within 45 min coincident with the maximal increases in circulating testosterone and estradiol levels. These events were followed by the emergence of an E₂-induced protein of Mr 27,000 at 3–6 h, and by reduction in the activity of 17α-hydroxylase/17–20 desmolase, and a decrease in microsomal cytochrome P-450. The negative effects of LH upon receptors and steroidogenic responses appear to be characteristic of the adult Leydig cell, and do not occur in the immature or fetal Leydig cell, where only up-regulation was demonstrated in vivo or in vitro. The temporal and functional nature of the steroidogenic lesions provide further insight into the intracellular control mechanisms that regulate the androgen biosynthetic pathways of the mature Leydig cell.     

The ins and outs of cytochrome P450s

The active site of cytochromes P450 is situated deep inside the protein next to the heme cofactor. Consequently, enzyme specificity and kinetics can be influenced by how substrates pass through the protein to access the active site and how products egress from the active site. We previously analysed the channels between the active site and the protein surface in P450 crystal structures available in October 2003 [R.C. Wade, P.J. Winn, I. Schlichting, Sudarko, A survey of active site access channels in cytochromes P450, J. Inorg. Biochem. 98 (2004) 1175-1182]. Since then, 52 new P450 structures have been made available, including entries for ten isozymes for which structures were not previously available. We present an updated survey covering all P450 crystal structures available in March 2006. This survey shows channels not observed earlier in crystal structures, some of which were identified in previous molecular dynamics simulations. The crystal structures demonstrate how some of the channels can merge when the protein structure opens up resulting in a wide cleft to the active site, caused largely by movements of the F-G helix-loop-helix and the B-C loop. Significant differences were observed between the channels in the crystal structures of the mammalian and bacterial enzymes. The multiplicity of channels suggests possibilities for substrate channelling to and from the P450s.     

Regulation of the biosynthesis of plant hormones by cytochrome P450s

 Cytochrome P450 monooxygenases are a large group of heme-containing enzymes, most of which catalyze hydroxylation reactions. Since the discovery of cytochrome P450 in plants, more than 500 forms have been found, and they appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites. In particular, cytochrome P450s are involved in the biosynthesis of plant hormones, and play important roles in the regulation of plant growth and development. Recent genetic and functional analyses of cytochrome P450s in plants have significantly improved our understanding of not only the biosynthetic pathways themselves, but also of plant development from the perspective of hormonal control of morphogenesis. This review summarizes the present status of research on cytochrome P450s' roles in regulating the biosynthesis of plant hormones. Received: January 30, 2002 / Accepted: March 4, 2002