Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.
Animals ; Escherichia coli/genetics* ; NADPH-Ferrihemoprotein Reductase ; Protein Engineering ; Rats ; Repressor Proteins ; Squalene ; Squalene Monooxygenase/genetics*

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.
Acetates ; Andrographis ; enzymology ; genetics ; Biosynthetic Pathways ; Cloning, Molecular ; Cyclopentanes ; Diterpenes ; metabolism ; NADPH-Ferrihemoprotein Reductase ; genetics ; Oxylipins ; Plant Leaves ; enzymology ; Plant Proteins ; genetics

Cyclophosphamide (CPA) is the most common alkylating antineoplastic agent, as well as a strong immunosuppressant that is frequently applied to autoimmune diseases and organ transplantation. It is metabolized by cytochrome P450 oxidases (CYPs) to its active metabolite which played a critical role in therapy. CPA has serious and even fatal side effects, and its efficacy and adverse reactions are significantly varied among individuals. In this review, the association of the genetic polymorphisms in the metabolic enzymes and transporters involved in the disposition of CPA with the efficacy and adverse effects of CPA were summarized, thereby providing fundamental reference for further pharmacogenomic study of CPA.
Antineoplastic Agents, Alkylating ; pharmacology ; Cyclophosphamide ; pharmacology ; Humans ; NADPH-Ferrihemoprotein Reductase ; metabolism ; Pharmacogenetics

Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.
Biosynthetic Pathways ; Biotransformation ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Diterpenes ; metabolism ; Diterpenes, Abietane ; biosynthesis ; chemistry ; Electrophoresis, Agar Gel ; Gene Amplification ; NADPH-Ferrihemoprotein Reductase ; genetics ; metabolism ; Open Reading Frames ; Plasmids ; Saccharomyces cerevisiae ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry

OBJECTIVETo investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells.

METHODSThe Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined.

RESULTSWhen the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min*g protein) and 4.34 ml/(min*g protein), respectively.

CONCLUSIONThe conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

Animals ; Cytochrome P-450 CYP3A ; biosynthesis ; Cytochromes b5 ; biosynthesis ; Humans ; Insecta ; NADPH-Ferrihemoprotein Reductase ; biosynthesis ; Sf9 Cells

CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.
Animals ; Baculoviridae ; enzymology ; genetics ; Catalysis ; Cells, Cultured ; Chromatography, High Pressure Liquid ; methods ; Cytochrome P-450 CYP2D6 ; genetics ; metabolism ; Dextromethorphan ; metabolism ; Isoenzymes ; metabolism ; NADPH-Ferrihemoprotein Reductase ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; Spodoptera ; cytology ; virology ; Transfection

Fatty acid binding and oxidation kinetics for wild type P450(BM3) (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k (cat). The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.
Bacterial Proteins ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Fatty Acids ; chemistry ; metabolism ; Lauric Acids ; chemistry ; metabolism ; Myristic Acid ; chemistry ; metabolism ; NADPH-Ferrihemoprotein Reductase ; metabolism ; Osmolar Concentration ; Oxidation-Reduction ; Palmitic Acid ; chemistry ; metabolism ; Structure-Activity Relationship

PURPOSE: To date, paraquat poisoning has almost universally resulted in unfavorable outcomes, and it has become a big issue in clinical toxicology. Current efforts to overcome its toxicity have focused on drugs with anti-oxidant capacity such as ascorbic acid in order to combat over-production of reactive oxygen species (ROS) by paraquat radicals, which are mainly induced by NADPH-cytochrome P450 reductase. Unfortunately, this strategy of treatment has not yielded satisfactory results. In search of a new approach to cope with PQ toxicity, we developed an in vitro culture model of cells resistant to lethal doses of PQ, and we then investigated resistance mechanisms using DNA microarray technology, a tool for simultaneously measuring a number of gene expression changes. METHODS: This experiment was conducted in vitro using the hepatocelluar carcinoma cell line (HepG2) to assay xenobitotics metabolism. We induced resistant of these cells to up to 100 uM PQ by treating with escalating doses of PQ for about 5 months. Cytotoxicity was studied using the MTT method, and optical density was measured at 540 nm using an ELISA reader. We examined morphological changes in cells after drug treatment using an inverted microscope, and we investigated gene expression profiles in control and resistant cells by use of DNA microarray. RESULTS: Results of MTT assays indicated that resistant cells showed relatively high survivals against a 100 mM dose, but that the control group had zero percents of survival at a 1 mM dose. In the comparing gene expression levels between the control group and the resistant group, 6,717 genes found to be differentially expressed. In the analysis of anti-apoptosis genes in particular, the resistant group showed more expression of genes with anti-apoptotic functions than did the control group. In examining the expression of cytochrome P450 genes related to xenobiotic metabolism and PQ radical induction, expression of the cytochrome P450 1B1 gene was significantly higher in the resistant group than in the control group. CONCLUSION: Although cytochrome P450 is known to be responsible for redox cycling of PQ as an electron transferor, this study suggest that up-regulation of the cytochrome P450 1B1 gene can corelate with PQ resistance. Therefore, induction of cytochrome P450 1B1 can be a new therapeutic approach to reduce PQ toxicity through actual PQ degradation, rather than simply through neutralization of ROS.
Ascorbic Acid ; Cell Line ; Cytochrome P-450 Enzyme System ; DNA ; Electrons ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hep G2 Cells ; NADPH-Ferrihemoprotein Reductase ; Oligonucleotide Array Sequence Analysis ; Oxidation-Reduction ; Paraquat ; Phosphatidylethanolamines ; Reactive Oxygen Species ; Toxicology ; Transcriptome ; Up-Regulation

Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has antioxidant effects. Here we aimed to determine the mechanism of actions of DHEA, in comparison to vitamin E, in a chemically-induced hepatocellular carcinoma model in rats. Sprague-Dawley rats were administered with control diet without a carcinogen, diets with 1.5% vitamin E, 0.5% DHEA and both of the compounds with a carcinogen for 6 weeks. The doses were previously reported to have anti-cancer effects in animals without known toxicities. With DHEA treatment, cytosolic malate dehydrogenase activities were significantly increased by ~5 fold and glucose 6-phosphate dehydrogenase activities were decreased by ~25% compared to carcinogen treated group. Activities of Se-glutathione peroxidase in the cytotol was decreased significantly with DHEA treatment, confirming its antioxidative effect. However, liver microsomal cytochrome P-450 content and NADPH-dependent cytochrome P-450 reductase activities were not altered with DHEA treatment. Vitamin E treatment decreased cytosolic Se-glutathione peroxidase activities in accordance with our previous reports. However, vitamin E did not alter glucose 6-phosphate dehydrogenase or malate dehydrogenase activities. Our results suggest that DHEA may have decreased tumor nodule formation and reduced lipid peroxidation as previously reported, possibly by increasing the production of NADPH, a reducing equivalent for NADPH-dependent antioxidant enzymes. DHEA treatment tended to reduce glucose 6-phosphate dehydrogenase activities, which may have resulted in limited supply for de novo synthesis of DNA via inhibiting the hexose monophophaste pathway. Although both DHEA and vitamin E effectively reduced preneoplastic foci in this model, they seemed to function in different mechanisms. In conclusion, DHEA may be used to reduce hepatocellular carcinoma growth by targeting NADPH synthesis, cell proliferation and anti-oxidant enzyme activities during tumor growth.
Animals ; Antioxidants ; Carcinoma, Hepatocellular ; Cardiovascular Diseases ; Cell Proliferation ; Chronic Disease ; Cytochrome P-450 Enzyme System ; Cytosol ; Dehydroepiandrosterone ; Diet ; DNA ; Glucose ; Glutathione Transferase ; Insulin Resistance ; Lipid Peroxidation ; Liver ; Malate Dehydrogenase ; Malates ; NADP ; NADPH-Ferrihemoprotein Reductase ; Oxidoreductases ; Peroxidase ; Rats ; Rats, Sprague-Dawley ; Vitamin E ; Vitamins

OBJECTIVETo investigate the effects of the ethyl acetate extract of Semen Hoveniae (ESH) on liver microsomal cytochrome P450 isoenzyme in rats.

METHODThe rats were given orally the ESH in the doses of 0.14, 0.17, 0.2 g x kg (equivalent to the crude herb) for 10 days respectively. Rat liver microsomal cytochrome P450, NADPH-Cyt C reductase, erythromycin N-demethylase (ERD), Aniline hydroxylase (ANH), aminopyrine N-demethylase (ADM) activities were quantitated by UV chromatography. The levels of mRNA expression of CYP1A1, CYP2C11, CYP2E1 and CYP3A1 were detected by semi-quantitative reverse transcripatase-polymerase chain reaction (RT-PCR).

RESULTThe cytochrome P450 content, NADPH-Cyt C reductase activities and erythromycin N-demethylase (ERD) activities were not affected. Aniline hydroxylase (ANH) activities in liver were decreased by up to35.1%; aminopyrine N-demethylase (ADM) activitiesin liver were increased by up to 42.4%. The mRNA expression of CYP1A1, CYP2C11 and CYP3A1 were found to be increased markedly.

CONCLUSIONA specific effect of ESH on liver microsomal cytochrome P450 isoenzyme in rats was observed in this investigation. ESH had various effects on liver microsomal cytochrome P450 isoenzyme.

Acetates ; chemistry ; Aminopyrine N-Demethylase ; metabolism ; Aniline Hydroxylase ; genetics ; metabolism ; Animals ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Cytochrome P-450 CYP1A1 ; genetics ; metabolism ; Cytochrome P-450 CYP2E1 ; genetics ; metabolism ; Cytochrome P-450 CYP3A ; genetics ; metabolism ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Cytochrome P450 Family 2 ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Male ; Microsomes, Liver ; drug effects ; enzymology ; NADPH-Ferrihemoprotein Reductase ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Rhamnaceae ; chemistry ; Seeds ; chemistry ; Steroid 16-alpha-Hydroxylase ; genetics ; metabolism