17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA.

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.     

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β-estradiol (E₂) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERα, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E₂ metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998. © 1998 Wiley-Liss, Inc.     

Association of functionally important polymorphisms in cytochrome P4501B1 with lung cancer

In the present study, genotype and haplotype frequencies of four polymorphisms of cytochrome P450 1B1 (CYP1B1) that cause amino acid changes (Arg-Gly at codon 48, Ala-Ser at codon 119, Leu-Val at 432 and Asn-Ser at codon 453) were studied in 200 patients suffering from lung cancer and equal number of controls. A significant difference was observed for the distribution of variant genotypes of CYP1B1Arg48Gly and Ala119Ser polymorphisms (CYP1B1*2) in cases when compared to the controls. No significant difference was observed for the distribution of variant genotypes of CYP1B1Leu432Val (CYP1B1*3) and CYP1B1Asn453Ser (CYP1B1*4) polymorphism. When the four SNPs were analyzed using a haplotype approach, SNPs at codon 48 (Arg48Gly) and codon 119 (Ala119Ser) exhibited complete linkage disequilibrium (LD) in all the cases and controls. Significant differences in the distribution of the three haplotypes (G-T-C-A, G-T-G-A and G-T-C-G) were observed in the cases when compared to controls. Tobacco use in the form of smoking as well as chewing was found to significantly increase the risk of lung cancer in patients by interacting with CYP1B1Ala119Ser genotypes demonstrating the role of gene-environment interaction in lung cancer. Further, the risk of lung cancer increased several fold in the patients carrying the genotype combinations of CYP1B1Ala119Ser and CYP1B1Leu432Val with GSTM1, a phase II enzyme suggesting the importance of gene-gene interactions in enhancing the susceptibility to lung cancer.     

The effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a novel 17β-estradiol 4-hydroxylase

Rates of microsomal 17β-estradiol (E₂) hydroxylation at the C-2, -4, -6, and -15 positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 and -15 positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low K_m 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E₂ catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low K_m hydroxylation of E₂ at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE₂) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE₂ formation predominantly over 4-MeOE₂. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E₂ 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.     

Peroxide-supported in-vitro cytochrome P450 activities in Haemonchus contortus.

The potential for cytochrome P450 from Haemonchus contortus to operate in the oxygen-poor intestinal environment was investigated by examining the ability of the cytochrome to act in vitro as a peroxygenase in utilising cumene hydroperoxide for substrate oxidations not requiring molecular oxygen. Peroxygenase and NADPH-supported monooxygenase activities were measured in microsomes prepared from L3 and adult nematodes. Both cumene hydroperoxide- and NADPH-supported ethoxycoumarin O-deethylase and aldrin epoxidase activities were detected in larval microsomes. Adult microsomes showed low levels of cumene hydroperoxide-supported ethoxycoumarin O-deethylase, as well as NADPH- and cumene hydroperoxide-supported aldrin epoxidase activities. The use of inhibitors in ethoxycoumarin O-deethylase assays with larval microsomes indicated that the peroxygenase pathway does not proceed via ferrous cytochrome P450 (no inhibition by carbon monoxide), did not require molecular oxygen, and did not depend on electron flow from cytochrome P450 reductase. Larval activity was inhibited by typical cytochrome P450 inhibitors (piperonyl butoxide, SKF-525A, chloramphenicol, metyrapone, n-octylamine) and was unaffected by the peroxidase inhibitor salicylhydroxamic acid. In contrast, adult microsomal cumene hydroperoxide-supported ethoxycoumarin O-deethylase activity was significantly inhibited by both cytochrome P450 inhibitors and salicylhydroxamic acid. Adult microsomes also contained potassium ferrocyanide peroxidase activity utilising cumene hydroperoxide. This activity showed a similar pattern of inhibition by both cytochrome P450 and peroxidase inhibitors. Whilst the ability of larval H. contortus cytochrome P450 to act as a peroxygenase in vitro was demonstrated, the inhibition results with adult microsomes showing both cytochrome P450 and peroxidase activities require further investigation to clarify the nature of the adult microsomal cumene hydroperoxide-supported O-deethylase activity.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.     

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.     

A rational approach to Re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5

The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.     

Polymorphisms of the CYP1B1 gene have higher risk for prostate cancer

Various carcinogenic factors including estrogen metabolites play a role in malignant transformation. These metabolites are formed in part, as a result of the hydroxylation activity of cytochrome P450 (CYP) 1B1. Variant forms of this enzyme have been shown to enhance its activity, and thus, we hypothesize that single nucleotide polymorphisms of the CYP1B1 gene can be a risk factor for prostate cancer. To test this hypothesis, the genetic distribution of six different CYP1B1 polymorphisms at intron 1 (C-->T), codon 48 (C-->G), codon 119 (G-->T), codon 432 (C-->G), codon 449 (C-->T), and codon 453 (A-->G) was analyzed in 117 prostate cancer samples and 200 healthy normal subjects from a Japanese population. Results of these experiments demonstrate that the genotype at codon 119 is significantly different between prostate cancer patients and controls (P<0.001). The odds ratio of genotype T/T compared to G/G (reference) was calculated as 4.02 with a 95% confidence interval of 1.73-9.38. All other codons, except 453, showed polymorphisms but were not significantly different between cancer patients and controls. No association was found between stage and grade of cancer with any of the polymorphic sites. This is the first report that demonstrates the polymorphism at codon 119 of CYP1B1 to be associated with prostatic carcinogenesis. These results are important in understanding the role of CYP1B1 polymorphisms in the pathogenesis of prostate cancer.     

Omega- and (omega-1)-hydroxylation of lauric acid and arachidonic acid by rat renal cytochrome P-450

We resolved four cytochrome P-450s, designated as P450 K-2, K-3, K-4, and K-5, from the renal microsomes of untreated male rats by high-performance liquid chromatography (HPLC) and investigated the lauric acid and arachidonic acid hydroxylation activities of these fractions. P450 K-4 and K-5 had high omega- and (omega-1)-hydroxylation activities toward lauric acid. The ratio of the omega-/(omega-1)-hydroxylation activity of P450 K-4 and K-5 was 3 and 6, respectively. Also, P450 K-4 and K-5 effectively catalyzed the omega- and (omega-1)-hydroxylation of arachidonic acid. P450 K-3 was not efficient in the hydroxylation of either lauric acid or arachidonic acid. P450 K-2 had low omega- and (omega-1)-hydroxylation activities toward arachidonic acid, and efficiently catalyzed the hydroxylation of lauric acid at the (omega-1)-position only, not at the omega-position.     

CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime

The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.     

Bioengineering anabolic vitamin D-25-hydroxylase activity into the human vitamin D catabolic enzyme, cytochrome P450 CYP24A1, by a V391L mutation

CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) to different products: calcitroic acid or 1α,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D(3) (1α-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1α-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)(2)D(3) from 1α-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D(3) above the heme for hydroxylation.     

Identification of beta-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis, and subsequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450-dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor-inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (beta-amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean-12-ene-3beta,24-diol and soyasapogenol B, respectively, by GC-MS. Co-expression of CYP93E1 and beta-amyrin synthase in S. cerevisiae yielded olean-12-ene-3beta,24-diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a beta-amyrin and sophoradiol 24-hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene-monool to triterpene-triol.     

Highly sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes

A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.     

Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES

The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1alpha,25(OH)D3. Both 1alpha-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1alpha(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1alpha(OH)D3 were estimated to be 1.7 microM and 0.51 mol/min/mol P450, respectively, while those for 1alpha-hydroxylation activity toward 25(OH)D3 were 0.050 microM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1alpha-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1alpha,25(OH)D3, 1alpha-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.     

Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes

Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1, although no significant differences were statistically observed. The K(m) values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 microM for chlorzoxazone 6-hydroxylation, and 422 and 514 microM for 4-nitrophenol 2-hydroxylation, respectively. The K(m) values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. V(max) and V(max)/K(m) values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102%). In yeast cell microsomes expressing humCYP2E1, V(max) and V(max)/K(m) values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase

Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].