Pheophorbide Derivatives Isolated from Açaí Berries () Activate an Antioxidant Response Element .

Activity-guided fractionation was used to isolate and identify two components of the Brazilian açaí berry ( Mart.) with the ability to induce antioxidant response element (ARE)-dependent gene transcription in human hepatoma (HepG2) cells. Using an ARE-Luciferase reporter construct in cultured HepG2 cells, a suite of fractions from dried and powdered açaí berries were evaluated for transcriptional up-regulation of the luciferase gene.

High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants.

Epigenetic modifications enable cells to genetically respond to chemical inputs from environmental sources. These marks play a pivotal role in normal biological processes (e.g.

A dual substrate kinetic model for cytochrome P450-F87G catalysis: simultaneous binding of long chain aldehydes and 4-fluorophenol.

Objective: To develop a model for binding and catalysis associated with the stimulation of 4-fluorophenol (4-FP) oxidation in the presence of long chain aldehydes by the enzymatic catalyst, cytochrome P450-F87G.

Results: A variation of the Michaeli-Menten kinetic model was employed to describe interactions at the active site of the enzyme, along with computer aided modeling approaches. In addition to the hydroquinone product arising from de-fluorination of 4-FP, a second product (p-fluorocatechol) was also observed and, like the hydroquinone, its rate of formation increased in the presence of the aldehyde.

Inhibition of human cytochrome P450 2E1 and 2A6 by aldehydes: structure and activity relationships.

The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes.

Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: toward a functional halophenol bioremediation catalyst.

Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone, a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by Wiese et al.

Defluorination of 4-fluorophenol by cytochrome P450(BM₃)-F87G: activation by long chain fatty aldehydes.

Cytochrome P450(BM3)-F87G catalyzed the oxidative defluorination of 4-fluorophenol, followed by reduction of the resulting benzoquinone to hydroquinone via the NADPH P450-reductase activity of the enzyme. The k (cat) and K (m) for this reaction were 71 ± 5 min(-1) and 9.5 ± 1.

Isotopic labeling of the heme cofactor in cytochrome P450 and other heme proteins.

A recombinant bacterial expression system that generates (13)C-labeled heme or (15)N-labeled heme in functional cytochrome P450 enzymes and other heme-containing systems is reported here using a mutant strain of Escherichia coli (HU227) in which the HemA gene is inactive. By synthesizing several isotopomers of aminolevulinic acid with (13)C or (15)N at different locations, isotopes have been incorporated with high abundance into the heme cofactor of five different cytochrome P450 isoforms, along with one peroxidase. Confirmed both (13)C- and (15)N-incorporation; spectral and catalytic assays show the labeled enzymes produced in this system are functional.

The mechanism of oxidative halophenol dehalogenation by Amphitrite ornata dehaloperoxidase is initiated by H2O2 binding and involves two consecutive one-electron steps: role of ferryl intermediates.

The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species.

Effects of herbal products and their constituents on human cytochrome P450(2E1) activity.

Ethanolic extracts from fresh Echinacea purpurea and Spilanthes acmella and dried Hydrastis canadensis were examined with regard to their ability to inhibit cytochrome P450(2E1) mediated oxidation of p-nitrophenol in vitro. In addition, individual constituents of these extracts, including alkylamides from E. purpurea and S.

PksS from Bacillus subtilis is a cytochrome P450 involved in bacillaene metabolism.

As part of the pksX gene cluster of Bacillus subtilis strain 168, pksS has been preliminarily annotated as a cytochrome P450 homolog that hydroxylates the polyketide product of this cluster, which was recently shown to be involved in the biosynthesis of bacillaene and dihydrobacillaene. Here we report that there is a frame-shift error in the reported sequence for pksS, and that we have successfully cloned, overexpressed, and purified the protein encoded by the corrected sequence. By utilizing electronic absorption spectrophotometry, we have observed that the ferrous CO complex of PksS absorbs maximally near 450 nm, which confirms the annotation that this protein is a cytochrome P450.

Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors.

Rapid mixing of substrate-free ferric cytochrome P450(BM3)-F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450(CAM) and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme.

Liver enzyme-mediated oxidation of Echinacea purpurea alkylamides: production of novel metabolites and changes in immunomodulatory activity.

The medicinal plant Echinacea is widely used to treat upper respiratory infections and is reported to stimulate the human immune system. A major constituent class of Echinacea, the alkylamides, has immunomodulatory effects. Recent studies show that alkylamides are oxidized by cytochrome P450 enzymes, but the immunomodulatory activity of these products is unknown.

Effects of green tea extracts on gene expression in HepG2 and Cal-27 cells.

Green tea extract is known to contain compounds that are able to produce antioxidant effects in many types of living cells. Treatment of cultured human hepatoma (HepG2) cells with green tea extract resulted in dramatically increased expression of at least 15 genes that are present on a commercial human drug metabolism gene array. RT-PCR was used to confirm the microarray results, and analysis of the 5'-flanking region of each of these genes revealed potential electrophile/antioxidant response elements.

C. fumago chloroperoxidase is also a dehaloperoxidase: oxidative dehalogenation of halophenols.

We have examined the H2O2-dependent oxidative dehalogenation of 2,4,6-trihalophenols and p-halophenols catalyzed by Caldariomyces fumago chloroperoxidase (CCPO). CCPO is significantly more robust than other peroxidases and can function under harsher reaction conditions, and so its ability to dehalogenate halophenols could lead to its use as a bioremediation catalyst for aromatic dehalogenation reactions. Optimal catalysis occurred under acidic conditions (100 mM potassium phosphate solution, pH 3.

Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme.

We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 degrees C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM.

Cytochrome P450 expression and activities in human tongue cells and their modulation by green tea extract.

The expression, inducibility, and activities of several cytochrome P450 (CYP) enzymes were investigated in a human tongue carcinoma cell model, CAL 27, and compared with the human liver model HepG2 cells. The modulation effects of green tea on various CYP isoforms in both cell lines were also examined. RT-PCR analysis of CAL 27 cells demonstrated constitutive expression of mRNA for CYPs 1A1, 1A2, 2C, 2E1, 2D6, and 4F3.

Cytochrome P450 expression and activities in rat, rabbit and bovine tongue.

Xenobiotic metabolism in the tongue has received little attention in the literature. In the present study, we report a comparative analysis of constitutive cytochrome P450 (CYP) expression and activities in the tongue. First we compared catalytic activities of rabbit, rat and bovine tongue samples using the probe substrates 4-nitrophenol, 1-phenylethanol, caffeine and 7-ethoxycoumarin.

Regioselective peroxo-dependent heme alkylation in P450(BM3)-F87G by aromatic aldehydes: effects of alkylation on cataysis.

Cytochrome P450(BM3)-F87G reacts with aromatic aldehydes and hydrogen peroxide to generate covalent heme adducts in a reaction that may involve the formation of a stable isoporphyrin intermediate [Raner, G. M., Hatchell, A.

Farnesol as an inhibitor and substrate for rabbit liver microsomal P450 enzymes.

Farnesol and the related isoprenoids, geranylgeraniol, geranylgeranyl pyrophosphate, and farnesyl pyrophosphate, are produced in the endoplasmic reticulum of hepatocytes in mammals, and each serve important biological functions. Of these compounds, only farnesol was shown to significantly inhibit rabbit liver microsomal cytochrome P450 enzymes. The observed inhibition appeared to be reversible, and was not strictly competitive, but rather mixed in nature.