cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from <Emphasis Type="Italic">Coleus blumei</Emphasis> involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K _m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.     

Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/4-ketolase

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a β-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the β-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this β-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from β-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of β-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both β-ionone ends.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers

Summary The stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation).     

Identification of the proteins related to cytochrome P450 induced by fenvalerate in a <Emphasis Type="Italic">Trichoplusia ni</Emphasis> cell line

In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed. Our data showed that at the 0–15 μmol/L challenge concentration of fenvalerate, at which the vitality of Tn cells was not affected (p > 0.05), there was a tendency toward a dose- and time-response of total cellular P450s, which peaked at the 9 h (p < 0.05) and 12 h (p < 0.01) time points following 12.5 μmol/L stimulation with fenvalerate. The 2-DE assay detected more than 1300 protein spots in each two-dimensional gel, of which 33 spots displayed significant differences. Among the changed spots, three isoforms of P450 were identified. One of the three P450 cDNA fragments (CYP4L4) was cloned and sequenced, and its expression in treated Tn cells increased significantly (p < 0.01). It was found that fenvalerate induced the expression of P450s in insect cells. This suggests that fenvalerate could be metabolized by CYP4L4 through a hydroxylation reaction in insect cells.     

Physiological, biochemical, and genetic characterization of an alicyclic amine-degrading Mycobacterium sp. strain THO100 isolated from a morpholine-containing culture of activated sewage sludge

Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282^T. The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1’ were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450_mor system remained unaltered. In addition, we found a spontaneous deletion of P450_mor from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA.     

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

Mechanism of substitution of nonionic detergent Emulgen 913 for phospholipid as an activator of N-demethylase activity of cytochrome P450 form 2B4 (LM₂) has been studied. It is shown that such an activation takes place at the detergent concentrations below values critical for micelle formation. Under these conditions, Emulgen does not affect the hexameric state of the cytochrome. The stimulating effect proved to be similar in reconstituted monooxygenase systems containing (a) cytochrome P450 2B4 and NADPH-cytochrome P-450 reductase and (b) cytochrome 2B4 and organic hydroperoxides. These results indicate that the activation is due to an effect of the detergent upon P450 2B4 per se rather than upon P450/flavoprotein complex formation. The above conclusion is supported by the sedimentation data and measurement of the CD spectra of cytochrome P450 2B4 at 380–450 nm.     

Induction by alkaloids and phenobarbital of Family 4 Cytochrome P450s in Drosophila : evidence for involvement in host plant utilization

In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3^′ RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs. Received: 14 August 1997 / Accepted: 24 March 1998     

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH₂-terminal sequence and catalytic activity of them were determined. The NH₂-terminal sequences of six forms of cytochrome P-450 (designated P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome P-450 isozymes are of different molecular species. The hydrophobicity values of the NH₂-terminal sequences of P450 UT-1 and P450 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54 000, of the six forms of P-450. P450 UT-2 was active in demethylation of benzphetmaine, 450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-1 ad P450 UT-2 were active in the 2α- and 16α-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6β-, 7α- and 15α-hydroxylation of the same steroid. We believe that P450 UT-1, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P-450.     

Enhancement of Isoflavone Synthase Activity by Co-expression of P450 Reductase from Rice

Plant cytochrome P450s interact with a flavoprotein, NADPH-cytochrome P450 reductase (CPR), to transfer electrons from NADPH. The gene for rice P450 reductase (RCPR) was cloned and expressed in Saccaromyces cerevisiae, where the specific activity of the expressed RPCR was 0.91 U/mg protein. When isoflavone synthase gene (IFS) from red clover, used as a model system of plant cytochrome P450, was co-expressed with RCPR in yeast, the production of genistein from naringein increased about 4.3-fold, indicating that the RCPR efficiently interacts with cytochrome P450 to transfer electrons from NADPH.     

Inhibition of cytochrome P450 isozymes in rat liver microsomes by polysaccharides derived from Phellinus linteus

Polysaccharides (0.5, 1 and 3 mg ml^–1) from cultured broth and mycelia of Phellinus linteus inhibited cytochrome P450 (CYP) 1A1, CYP 1A2, CYP 2B1, and CYP 2E1 activities in rat liver microsomes. The polysaccharides from the broth of Phellinus linteus grown with 5% (v/v) mulberry extract had highest inhibitory potency for CYP 1A1, 1A2 and 2B1 activities. The most potent inhibitor of CYP 2E1 activity were the polysaccharides from the broth of Phellinus linteusgrown with 10% (v/v) mulberry extract.     

Tissue-specific expression of rice CYP72A21 induced by auxins and herbicides

Cytochrome monooxygenase P450s (CYPs) comprise one of the largest enzyme families in plants. Some P450s are involved in xenobiotic metabolism: they confer herbicide tolerance and are induced by chemical treatments. We isolated a novel P450 cDNA, CYP72A21 (accession number, AB237166), from rice (Oryza sativa L. cv. Nipponbare) seedlings treated with a mixture of 2,4-dichlorophenoxyacetic acid (2,4-D), chlorotoluron, phenobarbital, salicylic acid, and naphthalic anhydride (each 100 μM). We also isolated the gene’s promoter region. Endogenous CYP72A21 expression in rice seedlings treated with 2,4-D, herbicides esprocarb, or trifluralin was increased in the aerial part of seedlings. An expression plasmid, pI21pg, containing the GUS gene under the control of the CYP72A21 promoter was introduced into rice plants. GUS was expressed constitutively in roots, but this expression was suppressed by 2,4-D treatment. 2,4-D and other auxins induced GUS expression effectively in the stem and leaves. Histological observation revealed that GUS was expressed mainly in the base of the stem. Treatment with the herbicides acetochlor, esprocarb, and propyzamide induced GUS expression in the aerial parts of the seedlings. The CYP72A21 promoter was highly responsive to treatments with various chemicals, and thus might be useful for producing transgenic plants for biomonitoring of environmental chemicals.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Purification of hepatic microsomal cytochromes P-450 from β-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with β-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with β-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56 000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (M_r 55 000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identify with cod cytochrome P-450b (M_r 54 000), possibly as a result of contaminating cytochrome P-450c in this fraction.     

7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching,suggesting binding at multiple protein sites

Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern–Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (K_d 1.8–2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (K_d 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (K_d 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (K_d 0.048 and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.