Arabidopsis CYP85A2 Catalyzes Lactonization Reactions in the Biosynthesis of 2-Deoxy-7-oxalactone Brassinosteroids

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.     

Induction of Two Forms of Eel Cytochrome P450 1A Genes by 3-Methylcholanthrene

In eel (Anguilla japonica), exposure to polyaromatic hydrocarbons such as 3-methylcholanthrene leads to induction of two CYP1A enzymes, CYP1A1 and CYP1A6. We studied the time course and tissue specificity of induction of messenger RNAs for CYP1A1 and CYP1A6 in eel by administering 3-methylcholanthrene intraperitoneally. In both cases, the drug induced a rapid increase of mRNAs and biphasic expression. In the liver, mRNA levels of CYP1A1 and CYP1A6 increased 22-fold at 3 hours and 27-fold at 6 hours after the administration, respectively, showing initial peaks in the induction. After the initial inductions, mRNA levels decreased unexpectedly. Following these temporary decreases, the mRNA levels again increased and reached levels that were 35 and 41 times the basal levels at 24 hours after administration, respectively. CYP1A1 and CYP1A6 resembled each other also in the tissue specificity of gene expression; the expression levels were liver ≫ gill > intestine > kidney. The rapid induction, the biphasic expression, and the tissue-specific expression were common features of gene expression in CYP1A1 and CYP1A6 and may come from common structures of the regulatory regions of the two genes. Received December 7, 1998; accepted February 15, 1999     

Biocatalytic Synthesis of Dihydroxynaphthoic Acids by Cytochrome P450 CYP199A2

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was found to exhibit oxidation activity towards three hydroxynaphthoic acids. Whole cells of the recombinant Escherichia coli strain expressing CYP199A2 efficiently catalyzed the regioselective oxidation of 1-, 3-, and 6-hydroxy-2-naphthoic acids to produce 1,7-, 3,7-, and 6,7-dihydroxynaphthoic acid respectively. These results suggest that CYP199A2 might be a useful oxidation biocatalyst for the synthesis of dihydroxynaphthoic acids.     

CYP90A1/CPD, a Brassinosteroid Biosynthetic Cytochrome P450 of Arabidopsis, Catalyzes C-3 Oxidation

Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.     

Simultaneous measurement of CYP1A2 activity, regioselectivity, and coupling: Implications for environmental sensitivity of enzyme-substrate binding

The cytochrome P450 (CYP) reaction mechanism often yields a broad array of coupled and uncoupled products from a single substrate. While it is well known that reaction conditions can drastically affect the rate of P450 catalysis, their effects on regioselectivity and coupling are not well characterized. To investigate such effects, the CYP1A2 oxidation of 7-ethoxymethoxy-3-cyanocoumarin (EOMCC) was examined as a function of buffer type, buffer concentration, pH, and temperature. A high-throughput, optical method was developed to simultaneously measure the rate of substrate depletion, NADPH depletion, and generation of the O-dealkylated product. Increasing the phosphate buffer concentration and temperature increased both the NADPH and EOMCC depletion rates by 6-fold, whereas coupling was constant at 7.9% and the regioselectivity of O-dealkylation to other coupled pathways was constant at 21.7%. Varying the buffer type and pH increased NADPH depletion by 2.5-fold and EOMCC depletion by 3.5-fold; however, neither coupling nor regioselectivity was constant, with variations of 14.4% and 21.6%, respectively. Because the enzyme–substrate binding interaction is a primary determinant of both coupling and regioselectivity, it is reasonable to conclude that ionic strength, as varied by the buffer concentration, and temperature alter the rate without affecting binding while buffer type and pH alter both.     

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

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.     

Binding of bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine to wild-type and F120A mutant cytochrome P450 2D6 studied by resonance Raman spectroscopy

Cytochrome P450 2D6 (CYP2D6) is one of the most important drug-metabolizing enzymes in humans. Resonance Raman data, reported for the first time for CYP2D6, show that the CYP2D6 heme is found to be in a six-coordinated low-spin state in the absence of substrates, and it is perturbed to different extents by bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine (MDMA). Dextromethorphan and MDMA induce in CYP2D6 a significant amount of five-coordinated high-spin heme species and reduce the polarity of its heme-pocket, whereas bufuralol does not. Spectra of the F120A mutant CYP2D6 suggest that Phe120 is involved in substrate-binding of dextromethorphan and MDMA, being responsible for the spectral differences observed between these two compounds and bufuralol. These differences could be explained postulating a different substrate mobility for each compound in the CYP2D6 active site, consistently with the role previously suggested for Phe120 in binding dextromethorphan and MDMA.     

Polymorphisms of the Cytochrome P450 (CYP1A1, CYP2E1) and Microsomal Epoxide Hydrolase (mEPHX) Genes in Cystic Fibrosis and Chronic Respiratory Disease

Frequencies of CYP1A1, CYP2E1, and mEPHX polymorphic variants were analyzed in cystic fibrosis, chronic obstructive lung disease, bronchiectatic disease, chronic nonobstructive bronchitis, and recurring bronchitis. Mutations in CYP1A1 and mEPHX were shown to modify the severity of respiratory disorders in cystic fibrosis, the combination of CYP1A1 genotype Val/Val with the very slow mEPHX phenotype being most unfavorable (odds ratio OR = 12.30). Heterozygosity at both CYP1A1 and CYP2E1 was associated with chronic obstructive lung disease and recurring bronchitis (OR = 4.08 and 11.72, respectively). The very slow phenotype of mEPHX was predisposing to chronic respiratory disorders regardless of the CYP1A1 or CYP2E1 alleles (OR = 4.06). Basing on the above correlations, a combination of the very slow mEPHX phenotype with elevated cytochrome P450 (CYP1A1 and CYP2E1) activities was assumed to expedite severe respiratory disorders.     

Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation

Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val(117)--> Ala and Arg(372)--> His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala(117) --> Val and His(372) --> Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.     

Inhibition of benzo[a]pyrene-induced cytotoxicity and cytochrome P450 1A activity by dietary flavonoids in human liver cell model: structure-activity relationship

Inhibition of benzo[a]pyrene (B[a]P)-induced cytotoxicity and cytochrome p450 1A (CYP 1A) activity by flavonoids (1–100 M) was examined in terms of the structure-activity relationship in the human liver-derived cell model (HepG2). Two hydroxyl groups in the 5- and 7-position of flavonoids were essential to inhibit B[a]P-induced cytotoxicity. Generally, flavones (IC₅₀; 5.0–17.2 M) were more potent than the corresponding flavonols (IC₅₀; 42.7–131.8 M), and flavonoids such as apigenin (IC₅₀; 7.2 M) were more active than the corresponding isoflavonoids, genistein (IC₅₀; 61.7 M). The planar structure of flavone proved to be important in inhibiting B[a]P-induced toxicity and CYP 1A activity. The inhibitory effect of flavonoids on B[a]P-induced CYP 1A activity was correlated well with the inhibition of B[a]P-induced cytotoxicity (r=0.635, p<;0.01).     

Molecular cloning and functional analysis of cytochrome P450 1A2 from Japanese monkey liver: comparison with marmoset cytochrome P450 1A2

A cDNA encoding a novel cytochrome P450 1A2 (CYP1A2) was cloned from the liver of an adult female Japanese monkey. The CYP1A2 protein was expressed in yeast cells and its enzymatic properties were compared with those of marmoset CYP1A2 using ethoxyresorufin (ER) and phenacetin (PN) as substrates. The nucleotide sequence of Japanese monkey CYP1A2 revealed 94.7, 99.5 and 93.5% identities to those of human, cynomolgus monkey and marmoset monkey CYP1A2, respectively. Multiple amino acid sequence alignment of Japanese monkey CYP1A2 with CYP1A2 of humans, cynomolgus monkeys and marmosets showed that Japanese monkey CYP1A2 had 92.4, 99.0 and 91.9% identities to the human, cynomolgus monkey and marmoset enzymes, respectively. Kinetic studies demonstrated that the enzymatic properties as ER and PN O-deethylases were considerably different between the Japanese monkey and the marmoset CYP1A2. Furthermore, both of these reactions in liver microsomal fractions from the Japanese monkey and marmoset showed biphasic kinetics. On the basis of the kinetic parameters, it is suggested that Japanese monkey CYP1A2 is a high-K(m) enzyme in both ER and PN O-deethylations, whereas marmoset CYP1A2 is a high-K(m) and low-K(m) enzyme in ER and PN O-deethylations, respectively. alpha-Naphthoflavone, an inhibitor of human CYP1A1 and CYP1A2, did not completely inhibit the liver microsomal oxidations of ER and PN even at the highest concentration (50muM), supporting the notion that CYP1A2 enzymes are not the sole ER or PN O-deethylase in Japanese monkey and marmoset liver microsomes. Inhibitory effects of furafylline, an inhibitor of human CYP1A2, on ER O-deethylation by recombinant CYP1A2 enzymes were much lower than those of alpha-naphthoflavone, but marmoset CYP1A2 was more sensitive to furafylline than Japanese monkey CYP1A2. These results indicate that the properties of Japanese monkey CYP1A2 are considerably different from those of marmoset CYP1A2.     

Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116–134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.     

The signal-anchor sequence of CYP2C1 inserts into the membrane as a hairpin structure

Microsomal cytochrome P450s (CYPs) are anchored to the endoplasmic reticulum membrane by the N-terminal signal-anchor sequence which is predicted to insert into the membrane as a type 1 transmembrane helix with a luminally located N-terminus. We have mapped amino acids of the CYP2C1 signal-anchor, fused to Cys-free glutathione S-transferase, within the membrane by Cys-specific labeling with membrane-impermeant maleimide polyethylene glycol. At the C-terminal end of the signal-anchor, Trp-20 was mapped to the membrane–cytosol interface and Leu-19 was within the membrane. Unexpectedly, at the N-terminal end, Glu-2 and Pro-3 were mapped to the cytoplasmic side of the membrane rather than the luminal side as expected of a type 1 transmembrane helix. Similar results were observed for the N-terminal amino acids of the signal-anchor sequences of CYP3A4 and CYP2E1. These observations indicate that contrary to the current model of the signal-anchor of CYPs as a type 1 transmembrane helix, CYP2C1, CYP2E1, and CYP3A4 are monotopic membrane proteins with N-terminal signal-anchors that have a hairpin or wedge orientation in the membrane.     

The Aryl Hydrocarbon Receptor-interacting Protein (AIP) Is Required for Dioxin-induced Hepatotoxicity but Not for the Induction of the Cyp1a1 and Cyp1a2 Genes

The aryl hydrocarbon receptor (AHR) plays an essential role in the toxic response to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), in the adaptive up-regulation of xenobiotic metabolizing enzymes, and in hepatic vascular development. In our model of AHR signaling, the receptor is found in a cytosolic complex with a number of molecular chaperones, including Hsp90, p23, and the aryl hydrocarbon receptor-interacting protein (AIP), also known as ARA9 and XAP2. To understand the role of AIP in adaptive and toxic aspects of AHR signaling, we generated a conditional mouse model where the Aip locus can be deleted in hepatocytes. Using this model, we demonstrate two important roles for the AIP protein in AHR biology. (i) The expression of AIP in hepatocytes is essential to maintain high levels of functional cytosolic AHR protein in the mammalian liver. (ii) Expression of the AIP protein is essential for dioxin-induced hepatotoxicity. Interestingly, classical AHR-driven genes show differential dependence on AIP expression. The Cyp1b1 and Ahrr genes require AIP expression for normal up-regulation by dioxin, whereas Cyp1a1 and Cyp1a2 do not. This differential dependence on AIP provides evidence that the mammalian genome contains more than one class of AHR-responsive genes and suggests that a search for AIP-dependent, AHR-responsive genes may guide us to the targets of the dioxin-induced hepatotoxicity.     

A comparison of substrate dynamics in human CYP2E1 and CYP2A6

Considering the dynamic nature of CYPs, methods that reveal information about substrate and enzyme dynamics are necessary to generate predictive models. To compare substrate dynamics in CYP2E1 and CYP2A6, intramolecular isotope effect experiments were conducted, using deuterium labeled substrates: o-xylene, m-xylene, p-xylene, 2,6-dimethylnaphthalene, and 4,4'-dimethylbiphenyl. Competitive intermolecular experiments were also conducted using d(0)- and d(6)-labeled p-xylene. Both CYP2E1 and CYP2A6 displayed full isotope effect expression for o-xylene oxidation and almost complete suppression for dimethylbiphenyl. Interestingly, (k(H)/k(D))(obs) for d(3)-p-xylene oxidation ((k(H)/k(D))(obs)=6.04 and (k(H)/k(D))(obs)=5.53 for CYP2E1 and CYP2A6, respectively) was only slightly higher than (k(H)/k(D))(obs) for d(3)-dimethylnaphthalene ((k(H)/k(D))(obs)=5.50 and (k(H)/k(D))(obs)=4.96, respectively). One explanation is that in some instances (k(H)/k(D))(obs) values are generated by the presence of two substrates-bound simultaneously to the CYP. Speculatively, if this explanation is valid, then intramolecular isotope effect experiments should be useful in the mechanistic investigation of P450 cooperativity.     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.     

Differential in vivo effects of α‐naphthoflavone and β‐naphthoflavone on CYP1A1 and CYP2E1 in rat liver,lung, heart,and kidney

Male Sprague–Dawley rats were treated intraperitoneally with corn oil, the aryl hydrocarbon receptor (AHR) agonist β‐naphthoflavone (βNF), or the relatively weak AHR agonist α‐naphthoflavone (αNF). Animals treated with βNF experienced a significant loss (12%) of total body mass over 5 days and a dramatic elevation of CYP1A1 mRNA in all of the organs studied. Treatment with αNF had no significant effect on body mass after 5 days and caused only minor increases of liver, kidney, and heart CYP1A1 mRNA. In contrast, lung CYP1A1 mRNA was increased by αNF treatment to levels comparable to that seen with βNF treatment. CYP2E1 mRNA levels were also elevated in liver, lung, kidney, and heart in response to βNF treatment, whereas αNF was without effect. Large increases of CYP1A1‐dependent 7‐ethoxyresorufin O‐deethylation (EROD) activity occurred with microsomes prepared from the tissues of βNF‐treated animals. Comparatively small changes were associated with αNF treatment, with the exception of lung, where EROD activity was increased to approximately 60% of that with βNF treatment. CYP2E1‐dependent p‐nitrophenol hydroxylase (PNP) activity was also increased by βNF treatment in microsomes prepared from kidney (3.1‐fold), whereas αNF was without effect. In contrast, αNF or βNF treatment caused significant decreases of lung microsomal PNP (72% and 27% of corn oil control, respectively) and 7‐pentoxyresorufin O‐deethylation (48% and 17% of corn oil control, respectively) activities, indicating that PNP activity may be catalyzed by P450 isoforms other than CYP2E1 in rat lung. We conclude that βNF and αNF have differential effects on the expression and catalytic activity of CYP1A1 and CYP2E1, depending upon the organ studied. These changes most likely occur as a result of the direct actions of these compounds as AHR agonists, in addition to secondary effects associated with AHR‐mediated toxicity. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 29–40, 1999     

CYP1A1 gene polymorphisms and smoking status as modifier factors for lung cancer risk

Objective

Lung cancer remains the most prevalent malignancy worldwide. Susceptibility to lung cancer has been shown to be modulated by inheritance of polymorphic genes. Several metabolic enzymes are currently under investigation for their possible role in lung cancer susceptibility, including members of the cytochrome P450 (CYP) superfamily. The aim of this work was to identify the correlation between CYP1A1 m1 and m2 polymorphisms and lung cancer risk and figure its interactions with smoking as genetic modifiers in the etiology of lung cancer in the Egyptian population.

Materials and methods

One hundred and ten patients with lung cancer and one hundred and ten controls were enrolled in the study. CYP1A1 m1 and m2 polymorphisms were determined using polymerase chain reaction restriction fragment length polymorphism.

Results

Subjects carrying TC and CC genotypes of CYP1A1 m1 and AG and GG genotypes of CYP1A1 m2 were significantly more likely to develop lung cancer especially squamous cell carcinoma. The proportion of lung cancer attributable to the interaction of smoking and CYP1A1 m1 and CYP1A1 m2 polymorphisms was 32% and 52% respectively.

Conclusion

Our results revealed that CYP1A1 m1 and m2 polymorphisms contribute to smoking related lung cancer risk in the Egyptian population.