Distinct Cytochrome P450 Research Articles

Efficient O-demethylation of lignin monoaromatics using the peroxygenase activity of cytochrome P450 enzymes.

A crucial reaction in harnessing renewable carbon from lignin is O-demethylation. We demonstrate the selective O-demethylation of syringol and guaiacol using different cytochrome P450 enzymes. These can efficiently use hydrogen peroxide which, when compared to nicotinamide cofactor-dependent monooxygenases and synthetic methods, allows for cheap and clean O-demethylation of lignin-derived aromatics.

Cover Feature: N‐Heterocyclization in Gliotoxin Biosynthesis is Catalyzed by a Distinct Cytochrome P450 Monooxygenase (ChemBioChem 2/2021)

The cover feature picture shows a pop-art illustration of the pathogenic fungus Aspergillus fumigatus, which produces the infamous toxin, gliotoxin. Using targeted gene deletion, in silico studies and in vitro enzyme assays, we could elucidate a key late-stage step in the biosynthesis of gliotoxin. A single cytochrome P450 monooxygenase, GliF, is capable of catalyzing a N-heterocyclization reaction to form the hydroindole ring scaffold of gliotoxin. More information can be found in the communication by D. H. Scharf, C. Hertweck, et al.

N-Heterocyclization in Gliotoxin Biosynthesis is Catalyzed by a Distinct Cytochrome P450 Monooxygenase.

Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human‐pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways.

Visualization and isolation of zone-specific murine hepatocytes that maintain distinct cytochrome P450 oxidase expression in primary culture

Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/β-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.

Reconfigured Cyanogenic Glucoside Biosynthesis in Eucalyptus cladocalyx Involves a Cytochrome P450 CYP706C55.

Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are α-hydroxynitriles, and their hydrolysis releases toxic hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf nitrogen to the biosynthesis of the cyanogenic monoglucoside, prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in prunasin content, as well as the presence of the cyanogenic diglucoside amygdalin in flower buds and flowers. The identification and biochemical characterization of the prunasin biosynthetic enzymes revealed a unique enzyme configuration for prunasin production in E. cladocalyx This result indicates that a multifunctional cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of l-phenylalanine into its corresponding aldoxime, phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of phenylacetaldoxime to the α-hydroxynitrile, mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the dehydration of phenylacetaldoxime, an unusual CYP reaction. The resulting phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form mandelonitrile. The final glucosylation step to yield prunasin is catalyzed by a UDP-glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic glucosides in plants.

Construction and verification of CYP3A5 gene polymorphisms using a Saccharomycescerevisiae expression system to predict drug metabolism.

The present study evaluated the ability of a Saccharomyces cerevisiae expression system to predict the pharmacokinetic (PK) activity of a calcium channel blocker in patients with distinct cytochrome P450 3A5 (CYP3A5) polymorphisms. The blood pressure lowering activity of amlodipine in 57 hypertensive patients with CYP3A5*1/*1, CYP3A5*1/*3, CYP3A5*4 and CYP3A5*6 polymorphisms was evaluated by the current study. Subsequently, a Saccharomyces cerevisiae expression system for CYP3A5 gene polymorphisms was constructed to examine the PK activity of CYP3A5*1/*1, CYP3A5*4 and CYP3A5*6 polymorphisms. This system was used to predict the PK of amlodipine and was compared with the in vivo data from different gene polymorphism groups. The blood pressure lowering activity of amlodipine in hypertensive patients varied among CYP3A5 polymorphisms. The in vivo results demonstrated that CYP3A5*6 exhibited the highest metabolic rate, followed by CYP3A5*1/*1, CYP3A5*4 and CYP3A5*1/*3. The difference between CYP3A5*6 and CYP3A5*1/*1 was not statistically significant (P=0.5). In accordance with in vivo data, CYP3A5*1/*1 exhibited the highest in vitro metabolic rate, followed by CYP3A5*6 and CYP3A5*4. With the exception of the comparison between CYP3A5*6 and CYP3A5*1/*1, polymorphisms exhibited statistically significant differences compared with CYP3A5*1/*1 (P<0.05). The Saccharomyces cerevisiae expression system may be a cost effective and potentially useful tool for assessing the PK activity of drugs that are metabolized by CYP3A5.

Two cytochrome P450 aromatase genes in the hermaphrodite ricefield eel Monopterus albus: mRNA expression during ovarian development and sex change

Previously, the ricefield eel (Monopterus albus) was speculated to have only one cytochrome p450 aromatase gene. In this study, however, the cDNAs encoding two distinct cytochrome p450 aromatases, cyp19a1a and cyp19a1b, were isolated. The genomic organizations of both cyp19 genes were conserved when compared with other teleosts. Northern blot detected an abundant expression of cyp19a1a in the ovary, and cyp19a1b in the hypothalamus. RT-PCR coupled with Southern blot showed that cyp19a1a was expressed predominantly in the gonads of both sexes, with higher levels in the ovary than testis, while cyp19a1b was expressed in all the tissues examined in the male, but only in the brain and pituitary in the female. The levels of cyp19a1a mRNA in the ovary were increased significantly during vitellogenesis, but decreased significantly at mature stage. The levels of cyp19a1b mRNA in the brain and pituitary did not vary significantly during vitellogenesis. As ovarian development shifted from vitellogenesis to maturation, the levels of cyp19a1b mRNA was decreased significantly in the brain, but increased significantly in the pituitary. During natural sex change from female to male, the levels of cyp19a1a mRNA in the gonad were significantly decreased. The levels of cyp19a1b mRNA in the hypothalamus were significantly increased at the early intersexual phase, whereas the expression levels in the pituitary were significantly decreased at the intersexual phases. Taken together, these results showed a novel sexual dimorphism of cyp19a1b mRNA tissue distribution, and both CYP19 genes were associated with the ovarian development and natural sex change of the ricefield eel.

Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): Sexual dimorphism and onset of ontogenic expression

Cytochrome P450 aromatase (CYP19) is a key enzyme in the steroidogenic pathway that catalyses the conversion of testosterone to estrogen, and therefore is thought to influence gonadal sex differentiation. In an effort to understand the role of this enzyme in ovarian differentiation, we isolated cDNA encoding the two distinct isoforms, ovarian and brain (termed cyp19a and cyp19b, respectively) of adult common carp, Cyprinus carpio. The cloned cDNA for cyp19a had an open reading frame (ORF) of 518 amino acid residues, in contrast to cyp19b with an ORF of 511 amino acids. Sequence and phylogenetic analysis showed that these CYP19 isoforms were orthologous with previously described cyp19a and cyp19b from other teleosts. Quantitative real-time PCR indicated that both isoforms are expressed in adult ovary and brain, with predominant expression of cyp19a in the ovary and cyp19b in the brain. The major aromatase expressing tissue was found to be the brain, with greatest cyp19b expression in the anterior quarter (telencephalon) in both sexes. The gonad showed sexually dimorphic expression of both genes and dimorphic expression of cyp19a was observed in the cerebellum and the liver. Ontogenic expression showed that only the ovarian aromatase transcript is inherited maternally, with lower expression observed through early larval development under warmer rearing conditions. The differential and overlapping expression suggests these two aromatase genes have different roles in reproductive physiology.

Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol, 4-tert-octylphenol) modulate expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To understand the effect of endocrine-disrupting chemicals (EDCs) on cytochrome P450 aromatase ( rm- cyp19) gene expression between gender types in the hermaphroditic fish Rivulus marmoratus, we cloned two distinct rm- cyp19 genes using RT-PCR with degenerative primers, obtained full-length cDNAs using 5′- and 3′-RACE-PCR methods, and completely sequenced them. The brain aromatase ( rm- cyp19b) cDNA consisted of 2,124 bp including the open reading frame (ORF), which encoded a putative protein of 505 amino acids. The ovarian aromatase ( rm-cyp19a) cDNA consisted of 2,075 bp, including the ORF encoding a putative protein of 516 amino acids. Expression patterns of rm- cyp19b and rm-cyp19a mRNAs were investigated in embryos of different developmental stages and in seven different tissues of adult fish. The rm- cyp19b gene in hermaphrodite and secondary male R. marmoratus was predominantly expressed in the brain, while the rm- cyp19a gene was expressed gender-specifically in the gonad. The expression of rm- cyp19b mRNA increased from stage 1 (2d post fertilization) to stage 4 (12d post fertilization) in a developmental stage-dependent manner but steeply decreased in the hatching stage. Compared to the rm- cyp19b gene, the abundance of ovarian aromatase rm- cyp19a transcripts was very low, and its expression was first detected at stage 3 and then decreased gradually to the hatching stage. Alteration of rm- cyp19b and rm- cyp19a gene expression was further analyzed in the brain and gonad by real-time RT-PCR 96 h after EDC exposure in hermaphrodites and secondary males. The brain aromatase rm- cyp19b gene was up-regulated in the brain after 4-nonylphenol (4-NP)-exposure, while the ovarian aromatase rm- cyp19a gene was significantly down-regulated in the gonad. In 300 μg/L 4-tert octylphenol (4-tert-OP), or 600 μg/L bisphenol A-exposed brain and gonad, both rm- cyp19b and rm- cyp19a genes were up-regulated. In the case of secondary males, the rm- cyp19b gene was highly expressed in the 4-NP-exposed brain, while expression of the rm- cyp19a gene was not detected in the gonad. These results indicate that the expression of rm- cyp19a and rm- cyp19b genes is differently modulated according to estrogenic compounds and gender type of R. marmoratus.

Constitutive expression and localization of cytochrome P‐450 1A1 in rat and human brain: presence of a splice variant form in human brain1

Cytochrome P-450 function as mono-oxygenases and metabolize xenobiotics. CYP1A1, a cytochrome P-450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. Northern and immunoblot analyses revealed constitutive expression of Cyp1a1 and CYP1A1 in rat and human brain, respectively. CYP1A1 mRNA and protein were localized predominantly in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum and pyramidal neurons of CA1, CA2, and CA3 subfields of the hippocampus. RT-PCR analyses using RNA obtained from autopsy human brain samples demonstrated the presence of a splice variant having a deletion of 87 bp of exon 6. This splice variant was present in human brain, but not in the liver from the same individual, and was absent in rat brain and liver. Structural modeling indicated broadening of the substrate access channel in the brain variant. The study demonstrates the presence of a unique cytochrome P-450 enzyme in human brain that is generated by alternate splicing. The presence of distinct cytochrome P-450 enzymes in human brain that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics including drugs could occur in brain by pathways different from those known to occur in liver.

Two distinct cytochrome P450 aromatases in the orange-spotted grouper ( Epinephelus coioides): cDNA cloning and differential mRNA expression

The cDNA sequences encoding two distinct cytochrome P450 aromatases, namely P450aromB and P450aromA, were isolated from brain and ovary cDNA libraries of the orange-spotted grouper, respectively. The P450aromB cDNA consists of 1892 bp, and the open reading frame (ORF) encodes a putative protein of 506 amino acids. The P450aromA cDNA consists of 1836 bp, and the ORF encodes a putative protein of 518 amino acids. Northern blot analysis revealed a transcript of about 1.9 kb for P450aromB in the brain and kidney, and 2.1 kb for P450aromA in the ovary. The expression of both P450aromB and P450aromA genes in different tissues was further examined using one-step RT-PCR followed by Southern blot analysis. High levels of P450aromB mRNA expression were detected in the olfactory bulb, forebrain, midbrain, hypothalamus, medulla, pituitary, gill filament, gill arch, kidney, muscle, adipose tissue, and blood cells, but low levels in the hindbrain and ovary. High levels of P450aromA mRNA expression were detected in the ovary, pituitary, gill filament, gill arch, and spleen, but low levels in the forebrain, hindbrain, hypothalamus, and blood cells. In addition, the expression of P450arom genes in the orange-spotted grouper of different gonadal stages as induced by 17α-methyltestosterone (MT) was investigated. The mRNA expression of P450aromB in the hypothalamus was highest in the intersexual stage, whereas the mRNA expression of P450aromA in the gonads was highest in the female stage, decreased in the intersexual stage, and lowest in the male stage. Results from current study indicate that P450aromB and P450aromA genes of the orange-spotted grouper have distinct tissue patterns of mRNA expression, and both of them may be involved in the MT-induced sex change.

Developmental changes in the liver weight- and body weight-normalized clearance of theophylline, phenytoin and cyclosporine in children.

Body weight- (BW) normalized pediatric dosages of metabolically eliminated drugs often exceed the corresponding adult values. We aimed to clarify whether such findings would be attributable either to an augmented hepatic drug-metabolizing activity or to a systematic bias introduced by adopting BW as a size standard of clearance. We chose 3 model drugs that are metabolized by distinct cytochrome P450 (CYP) isoforms (theophylline, phenytoin and cyclosporine for CYPIA2, CYP2C9/2C19 and CYP3A4, respectively). The MEDLINE database covering 1966 to May 2001, was searched for articles where systemic clearance oftheophylline or oral clearance of cyclosporine and Vmax/ Km of phenytoin were reported with demographic data of individual children. Liver weights (LWs) of children were estimated using the equation constructed based upon the autopsy data in literature, and body surface area (BSA) was calculated using a standard formula. Relationships between age and clearance of the 3 model drugs that were normalized against BW, LW and BSA were examined. The analysis was confined to the data obtained from children older than 1 year due to scarcity of data for infants and neonates. Relevant data were obtained from 24, 46 and 14 children for theophylline, phenytoin and cyclosporine, respectively. The development of LW lags behind that of BW but is almost identical to that of BSA. Thus, children had a greater LW/BW ratio than adults. The BW-normalized clearance of theophylline and Vmax/Km of phenytoin showed significantly (p < 0.01) negative correlations with age (r = -0.43 and -0.50, respectively) during childhood, whereas their LW- or BSA-normalized clearances were independent of age. While our analyses were made upon limited numbers of subjects and range of age, the results suggest that children appear to have an augmented BW-normalized clearance for drugs of which metabolism is dominated by the CYP1A2, CYP2C9 or CYP3A4 due mainly to a lagged development of BW than that of LW during childhood. BSA would serve as a practical alternative to LW for scaling adult dosage of metabolically eliminated drugs to children.

Distinct Cytochrome P450 Aromatase Isoforms in Zebrafish (Danio rerio) Brain and Ovary Are Differentially Programmed and Estrogen Regulated during Early Development

As a first step toward understanding estrogen’s role in neurodevelopment, a PCR cloning strategy was used to isolate complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms in adult zebrafish (Danio rerio) brain and ovary (termed P450aromB and P450aromA, respectively). Sequence and phylogenetic analysis showed that the zebrafish P450arom forms are orthologs of previously identified cyp19b and cyp19a genes in goldfish. On Northern blots, a single 4.4-kb transcript of the P450aromB subtype was identified in brain, and a 2.1-kb transcript of the P450aromA subtype in ovary, but RT-PCR showed a degree of overlapping expression. Both messenger RNA (mRNA) forms were detected in unfertilized eggs and 1.5 hpf (cleavage stage) embryos but declined by 12 hpf, indicating maternal transfer. A secondary rise in mRNAs between 12–24 hpf indicated the onset of embryonic cyp19b and -a transcription. Both mRNA species accumulated progressively to 120 hpf (early larval stage), but the relative magnitude and pattern of change was isoform specific. Estradiol (E2, 1 μm) advanced and amplified the developmentally programmed accumulation of P450aromB mRNA, and ICI164.384 decreased expressed levels, implying blockade of an endogenous estrogen mediated regulatory component. Conversely, E2 had no effect or decreased P450aromA mRNA. The early embryonic expression of P450aromB and P450aromA isoforms, and differences in developmental programming and estrogen regulation, imply independent regulatory mechanisms and unique functions during major morphogenetic and differentiative events.

Separation of three P450 isozymes from liver microsomes of gilthead seabream treated with ß‐NF and partial purification of cytochrome P4501A1

Three distinct cytochrome P450 isozymes, P4501A1, P4502B and an unidentified P450 isozyme were separated and isolated from beta-NF-treated gilthead seabream liver microsomes. Cytochrome P4501A1 was partially purified from beta-NF-treated gilthead seabream liver microsomes in the presence of detergents Emulgen 913 and cholate and protease inhibitors using two DEAE-cellulose, Porapak Q and two hydroxylapatite column chromatographies. The overall yield of purified P4501A1 was 1.2% with respect to microsomal total P450 with a specific content of 3 nmol/mg protein. The purified P4501A1 was characterized with respect to spectral, electrophoretic, biocatalytic and immunochemical properties, which are found to be similar to P4501A1s purified from other teleost species such as trout, scup, cod and perch suggesting that the P450 we have purified belongs to CYP1A1.

Molecular Cloning and Expression of Two Novel Avian Cytochrome P450 1A Enzymes Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Transcriptional regulation by the aryl hydrocarbon receptor, for which the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent ligand, leads in mammalian liver to the induction of genes for two distinct cytochrome P450 (CYP)1A enzymes, CYP1A1 and -1A2. Fish seem to have only one CYP1A enzyme. CYP1A enzymes have been regarded as injurious largely because of their ability to activate chemical carcinogens. We report here the cloning and sequencing of cDNAs for two catalytically distinct TCDD-induced CYP enzymes in chick embryo liver. One mediates classic CYP1A1 activities. The other has some -1A2-like activities and is also responsible for TCDD-induced arachidonic acid epoxygenation, a much more conspicuous effect in liver of chicks than of mammalian species. Amino acid sequence analysis shows that although each chick enzyme can be classified in the CYP1A family, both are more like CYP1A1 than -1A2, and neither can be said to be directly orthologous to CYP1A1 or -1A2. Phylogenetic analysis shows that the two chick enzymes form a separate branch in the CYP1A family tree distinct from mammalian CYP1A1 and -1A2 and from fish CYP1A enzymes. The findings suggest that CYP1A progenitors split into two CYP enzymes with some parallel functions independently in two evolutionary lines, evidence for convergent evolution in the CYP1A family. Northern analysis shows that the chick enzymes have a different tissue distribution from CYP1A1 and -1A2. Polymerase chain reaction and in situ hybridization data show that both chick enzymes are expressed in response to TCDD even before organ morphogenesis. The findings further suggest that beyond their role in activating carcinogens, CYP1A enzymes have conferred evolutionary and developmental advantages, perhaps as defenses in maintaining homeostatic responses to toxic chemicals.

Cytochrome P-450 mediates tissue-damaging hydroxyl radical formation during reoxygenation of the kidney.

Renal reperfusion injury results from oxygen radical generation. During reoxygenation of hypoxic kidney cells, xanthine oxidase produces superoxide radical, which eventuates in hydroxyl radical formation by the Fenton reaction. This reaction, catalyzed by transition metals such as iron, is particularly important because hydroxyl radical is highly reactive with a wide variety of biomolecules. We tested the hypothesis that this catalytic function is fostered by iron released from the heme moiety of cytochrome P-450. Primary cultures of rat proximal tubule epithelial cells studied in a subconfluent stage were subjected to 60 min of hypoxia and 30 min of reoxygenation. When cells were pretreated with one of three cytochrome P-450 inhibitors (piperonyl butoxide, cimetidine, or ketoconazole), lethal cell injury was attenuated. There was the expected increase in O2-. production during hypoxia/reoxygenation that cytochrome P-450 inhibitors did not prevent; on the other hand, inhibitors did prevent reoxygenation-induced hydroxyl radical formation. Analogously, the increase in catalytic iron (bleomycin-detectable iron) that accompanies hypoxia/reoxygenation did not occur in the presence of cytochrome P-450 inhibitors. In vivo studies confirmed a protective effect of cytochrome P-450 inhibition because glomerular filtration rate was better preserved in rats pretreated with cimetidine and then subjected to renal artery occlusion. In summary, several chemically distinct cytochrome P-450 inhibitors reduced iron release, and thereby, hydroxyl radical formation and reoxygenation-induced lethal cell injury, without inhibiting superoxide radical formation. We conclude that highly labile P-450 may act as an Fe-donating catalyst for Fenton reaction production of HO.-mediated reperfusion injury.

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.

Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate.

A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G + C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P-450: cytochrome P-450RR1 was induced by 2-ethoxyphenol, and cytochrome P-450RR2 was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (KS = 4.2 +/- 0.3 microM) and 2-methoxyphenol (KS = 2.0 +/- 0.1 microM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450RR2 by both 4-methoxybenzoate (KS = 2.1 +/- 0.1 microM) and 4-ethoxybenzoate (KS = 1.6 +/- 0.1 microM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-450RR2, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the O-dealkylation of their respective substrates. The respective products of the O-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers.

Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants

The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide.

Isolation of two distinct cytochromes P-45011 beta with aldosterone synthase activity from bovine adrenocortical mitochondria.

Two distinct forms of cytochrome P-45011 beta, with apparent molecular weights of 48,500 (48.5K) and 49,500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-450(11) beta (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-450(11) beta were capable of catalyzing aldosterone synthesis as well as the 11 beta- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-450(11) beta species exist in the adrenal cortex and participate in steroidogenesis.