Mixed colonies of Aspergillus niger and Aspergillus oryzae cooperatively degrading wheat bran.

In both natural and man-made environments, microorganisms live in mixed populations, while in laboratory conditions monocultures are mainly used. Microbial interactions are often described as antagonistic, but can also be neutral or cooperative, and are generally associated with a metabolic change of each partner and cause a change in the pattern of produced bioactive molecules. A. niger and A. oryzae are two filamentous fungi widely used in industry to produce various enzymes (e.g. pectinases, amylases) and metabolites (e.g. citric acid). The co-cultivation of these two fungi in wheat bran showed an equal distribution of the two strains forming mixed colonies with a broad range of carbohydrate active enzymes produced. This stable mixed microbial system seems suitable for subsequent commercial processes such as enzyme production. XlnR knock-out strains for both aspergilli were used to study the influence of plant cell wall degrading enzyme production on the fitness of the mixed culture. Microscopic observation correlated with quantitative PCR and proteomic data suggest that the XlnR Knock-out strain benefit from the release of sugars by the wild type strain to support its growth.

Characterization of the transcriptional regulation of the tarIJKL locus involved in ribitol-containing wall teichoic acid biosynthesis in Lactobacillus plantarum.

Lactobacillus plantarum strains produce either glycerol (Gro)- or ribitol (Rbo)-backbone wall teichoic acid (WTA) (Gro-WTA and Rbo-WTA, respectively). The strain WCFS1 has been shown to be able to activate the tarIJKL locus involved in Rbo-WTA synthesis when the tagD1F1F2 locus for Gro-WTA synthesis was mutated, resulting in switching of the native Gro-WTA into Rbo-WTA. Here, we identify a regulator involved in the WTA backbone alditol switching and activation of the tarIJKL locus. Promoter reporter assays of the tarI promoter (Ptar) demonstrated its activity in the Rbo-WTA-producing mutant derivative (ΔtagF1-2) but not in the parental strain WCFS1. An electrophoresis mobility shift assay using a Ptar nucleotide fragment showed that this fragment bound to Ptar-binding protein(s) in a cell-free extract of WCFS1. Three proteins were subsequently isolated using Ptar bound to magnetic beads. These proteins were isolated efficiently from the lysate of WCFS1 but not from the lysate of its ΔtagF1-2 derivative, and were identified as redox-sensitive transcription regulator (Lp_0725), catabolite control protein A (Lp_2256) and TetR family transcriptional regulator (Lp_1153). The role of these proteins in Ptar regulation was investigated by knockout mutagenesis, showing that the Δlp_1153 mutant expressed the tarI gene at a significantly higher level, supporting its role as a repressor of the tarIJKL locus. Notably, the Δlp_1153 mutation also led to reduced expression of the tagF1 gene. These results show that Lp_1153 is a regulatory factor that plays a role in WTA alditol switching in Lb. plantarum WCFS1 and we propose to rename this gene/protein wasR/WasR, for WTA alditol switch regulator.

A proteomic perspective on the changes in milk proteins due to high somatic cell count.

Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 10(5) to 10(6) cells/mL were analyzed qualitatively and quantitatively using both one-dimension sodium dodecyl sulfate PAGE and filter-aided sample preparation coupled with dimethyl labeling, both followed by liquid chromatography tandem mass spectrometry. Minor differences were found on the qualitative level in the proteome from milk with different SCC levels, whereas the concentration of milk proteins showed remarkable changes. Not only immune-related proteins (cathelicidins, IGK protein, CD59 molecule, complement regulatory protein, lactadherin), but also proteins with other biological functions (e.g., lipid metabolism: platelet glycoprotein 4, butyrophilin subfamily 1 member A1, perilipin-2) were significantly different in milk from cows with high SCC level compared with low SCC level. The increased concentration of protease inhibitors in the milk with higher SCC levels may suggest a protective role in the mammary gland against protease activity. Prostaglandin-H2 D-isomerase showed a linear relation with SCC, which was confirmed with an ELISA. However, the correlation coefficient was lower in individual cows compared with bulk milk. These results indicate that prostaglandin-H2 D-isomerase may be used as an indicator to evaluate bulk milk quality and thereby reduce the economic loss in the dairy industry. The results from this study reflect the biological phenomena occurring during subclinical mastitis and in addition provide a potential indicator for the detection of bulk milk with high SCC.

Trackability of proteins from probiotic Bifidobacterium spp. in the gut using metaproteomics.

Beneficial effects of Bifidobacterium spp. on gut microbiota development and infant health have been reported earlier. Therefore, supplementation of infant formulas with probiotic bifidobacteria, as well as prebiotics stimulating bifidobacterial growth, has been proposed. Here, we studied the faecal microbiome of infants supplemented with specialized nutrition, of which some received a standard amino acid-based formula (AAF) and others an AAF with a specific mixture of prebiotics and a probiotic (synbiotics) using metaproteomics and 16S rRNA gene sequencing. Faecal samples were taken at baseline, as well as after 6 and 12 months fed with the specialized formula. The aim was to compare microbial differences between infants treated with the standard AAF and those who received the additional synbiotics. Our findings show that infants who received AAF with synbiotics have significantly higher levels of Bifidobacteriaceae DNA as well as significantly increased levels of Coriobacteriaceae proteins, over time. Moreover, at visit 12 months higher levels of some bifidobacterial carbohydrate-active enzymes, known to metabolize oligosaccharides, were observed in the synbiotic group compared to the non-synbiotic group. The results indicate that the synbiotics provided in our study are trackable in faecal samples on the DNA and protein level during the intervention period.

A proteomics-based identification of putative biomarkers for disease in bovine milk.

The objective of this study was to identify and characterize potential biomarkers for disease resistance in bovine milk that can be used to indicate dairy cows at risk to develop future health problems. We selected high- and low-resistant cows i.e. cows that were less or more prone to develop diseases according to farmers' experience and notifications in the disease registration data. The protein composition of milk serum samples of these high- and low-resistant cows were compared using NanoLC-MS/MS. In total 78 proteins were identified and quantified of which 13 were significantly more abundant in low-resistant cows than high-resistant cows. Quantification of one of these proteins, lactoferrin (LF), by ELISA in a new and much larger set of full fat milk samples confirmed higher LF levels in low- versus high-resistant cows. These high- and low-resistant cows were selected based on comprehensive disease registration and milk recording data, and absence of disease for at least 4 weeks. Relating the experienced diseases to LF levels in milk showed that lameness was associated with higher LF levels in milk. Analysis of the prognostic value of LF showed that low-resistant cows with higher LF levels in milk had a higher risk of being culled within one year after testing than high-resistant cows. In conclusion, LF in milk are higher in low-resistant cows, are associated with lameness and may be a prognostic marker for risk of premature culling.

TEAC antioxidant activity of 4-hydroxybenzoates.

The influence of pH, intrinsic electron donating capacity, and intrinsic hydrogen atom donating capacity on the antioxidant potential of series of hydroxy and fluorine substituted 4-hydroxybenzoates was investigated experimentally and also on the basis of computer calculations. The pH-dependent behavior of the compounds in the TEAC assay revealed different antioxidant behavior of the nondissociated monoanionic form and the deprotonated dianionic form of the 4-hydroxybenzoates. Upon deprotonation the radical scavenging ability of the 4-hydroxybenzoates increases significantly. For mechanistic comparison a series of fluorobenzoates was synthesized and included in the studies. The fluorine substituents were shown to affect the proton and electron donating abilities of 4-hydroxybenzoate in the same way as hydroxyl substituents. In contrast, the fluorine substituents influenced the TEAC value and the hydrogen atom donating capacity of 4-hydroxybenzoate in a way different from the hydroxyl moieties. Comparison of these experimental data to computer-calculated characteristics indicates that the antioxidant behavior of the monoanionic forms of the 4-hydroxybenzoates is not determined by the tendency of the molecule to donate an electron, but by its ability to donate a hydrogen atom. Altogether, the results explain qualitatively and quantitatively how the number and position of OH moieties affect the antioxidant behavior of 4-hydroxybenzoates.

NADPH-cytochrome reductase catalysed redox cycling of 1,4-benzoquinone; hampered at physiological conditions, initiated at increased pH values.

In the present study the inability of 1,4-benzoquinone to support NADPH-cytochrome reductase catalysed redox cycling was investigated. The results obtained demonstrate that NADPH-cytochrome reductase is able to initiate a rapid two-electron reduction of 1,4-benzoquinone resulting in formation of the hydroquinone. The intermediate one-electron reduced semiquinone form does not pass its electron on to molecular oxygen, i.e. giving rise to redox cycling, but is reduced by a second electron, either by NADPH-cytochrome reductase upon protonation of the semiquinone or through disproportionation, both giving rise to the two-electron reduced hydroquinone. At pH values below the pK alpha of the hydroquinone, the electrons of the hydroquinone are also not passed on to molecular oxygen due to efficient protonation. However, at pH values around or above the pK alpha (9.85) of the two-electron reduced hydroquinone form, significant redox cycling activity is observed in a 1,4-benzoquinone containing incubation. Further experiments demonstrate a similarity in both the concentration and pH dependence of 1,4-benzoquinone or 1,4-hydroquinone supported NADPH-cytochrome reductase catalysed redox cycling. From these observations it is concluded that 1,4-benzoquinone is able to redox cycle from its deprotonated two-electron reduced hydroquinone form, but only at relatively high pH values. Together the data provide an insight into why the NADPH-cytochrome reductase catalysed redox cycling of 1,4-benzoquinone is inhibited at physiological conditions, but initiated at increased pH values.

MP8-dependent oxidative dehalogenation: evidence for the direct formation of 1,4-benzoquinone from 4-fluorophenol by a peroxidase-type of reaction pathway.

The present study shows that MP8 in the presence of H2O2 is able to catalyze the rupture of the stable carbon-fluorine bond of 4-fluorophenol, used as a model substrate for the oxidative dehalogenation reaction. 1,4-Benzoquinone was shown to be the primary reaction product. It is also demonstrated that there was significant [18O] incorporation into the product, 1,4-benzoquinone, from 18O-labelled H2(18)O but not from H2(18)O2. This implies that water participates in the reaction mechanism, and acts as a source for the oxygen atom inserted into the product. It also suggests that the reaction is not a result of direct oxygen transfer from H2O2 through the heme catalyst to the product. Furthermore, ascorbic acid, known to efficiently block MP8-catalyzed peroxidase-type conversions, inhibits the MP8-dependent dehalogenation reaction, most likely because of its ability to reduce the phenoxy radical back to the parent substrate. This observation together with the above-mentioned incorporation of oxygen from the solvent into the benzoquinone product indicates that MP8 dehalogenates 4-fluorophenol and converts it to 1,4-benzoquinone in a peroxidase- and not a P-450-type of reaction mechanism. Overall, our results indicate that the oxidative dehalo genation of para-halogenated phenols, resulting in the formation of benzoquinones, is not specific only for cytochrome P-450 enzymes. Hemoproteins exhibiting peroxidase activity could also play a role in the metabolism of these xenobiotics, resulting in the formation of electrophilic reactive benzoquinone type metabolites.

Interaction between the substrate and the high-valent-iron-oxo porphyrin cofactor as a possible factor influencing the regioselectivity of cytochrome P450 catalysed aromatic ring hydroxylation of 3-fluoro(methyl)anilines.

In the present study the in vitro and in vivo aromatic ring hydroxylation of a series of amino and/or methyl containing fluorobenzenes, i.e. 3-fluoro(methyl)anilines, was investigated and compared to the calculated density distribution of the reactive frontier pi-electrons of the aromatic substrate. This was done (1) to study to what extent the regioselectivity of the aromatic ring hydroxylation of the 3-fluoro(methyl)anilines could be predicted on the basis of the calculated chemical reactivity, as was previously observed for a series of fluorinated benzenes and monofluoroanilines, and (2) to investigate which factors contribute to possible deviations from the predictions on the basis of the calculated chemical reactivity. Results obtained show that the in vitro and in vivo aromatic ring hydroxylation of the series of 3-fluoro(methyl)anilines correlates qualitatively with the calculated frontier orbital density distribution for electrophilic attack by the cytochrome P450(FeO)3+ species. These results indicate that the HOMO/HOMO-1 frontier orbital densities, i.e. the chemical reactivity of the carbon centres for an electrophilic attack, predict the preferential as well as the non-reactive sites for cytochrome P450 catalysed aromatic ring hydroxylation of the tested model compounds. The absolute values, however, deviated in a systematic way; C4 para hydroxylation being observed to a higher extent than expected on the basis of chemical reactivity and C2/C6 ortho hydroxylation being observed to a lower extent than expected. Additional experiments were performed using different microsomal preparations and microperoxidase-8. The latter is a mini-heme protein of eight amino acids without a substrate binding site. In incubations of the model compounds with different types of microsomal preparations, as well as with MP-8 and purified reconstructed cytochrome P4502B1, similar systematic deviations between the predicted and observed regioselectivity of aromatic hydroxylation were observed. These results show that the regioselectivity of aromatic ring hydroxylation of the 3-fluoro(methyl)anilines cannot be predominantly ascribed to an interaction between the substrate and the substrate binding site of the cytochromes P450 dictating a specific stereoselective positioning of the substrate in the active site. More likely, the systematic deviations between the observed and predicted regioselectivity of hydroxylation of the tested model substrates should be ascribed to an (orienting) interaction between the substrate and the activated cytochrome P450(FeO)3+ cofactor.

Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as environmental factors. It is therefore important to study the qualitative and quantitative differences of bovine milk samples. Proteins in milk can be present in casein micelles, in the serum (the water soluble fraction) or in fat globules. These fat globules have a double membrane layer with proteins being bound to or being incapsulated in the membrane layer. The identification and molecular composition of the milk proteins have gained increased interest in recent years. Proteomic techniques make it now possible to identify up to many thousands of proteins in one sample, however quantification of proteins is as yet not straightforward. We analyzed the proteins of the milk fat globule membrane using dimethyl labeling methods combined with a filter-aided sample preparation protocol. Using these methods, it is now possible to quantitatively study the detailed protein composition of many milk samples in a short period of time.

Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism.

Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17ß-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.

Steroid conversions by Flavobacterium dehydrogenans in two-liquid-phase systems.

The production of 4-androstene-3, 17-dione by Flavobacterium dehydrogenans from androstenolone-acetate in octane/culture (v/v = 1:1) two-liquid phase systems was compared with the conversion rate in a Tween-containing medium commonly used in industry, and in a culture where no organic solvent or detergent was added. Variation of the cell density of the cultures at the moment of steroid, Tween, and/or organic solvent addition showed that the highest conversion rate in the three systems was reached with cells which were in the late exponential growth stage. The rate of 4-androstene-3, 17-dione production at the optimal cell density in the two-liquid-phase system and Tween medium were ca. 6 and 1(1/2) times as high, respectively, as in the aqueous medium without addition. The beneficial effect of octane on the rate of steroid conversion during growth could be explained by viability changes as measured by a new method to determine viability. Furthermore, our results show that a very high degree of conversion can be reached in the two-liquid-phase system [more than 98% (w/w)] which cannot be obtained in an aqueous medium. An easy way to recover the product in high purity is proposed.

Rules for optimization of biocatalysis in organic solvents.

General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.

Qualitative and quantitative influences of ortho chlorine substituents on the microsomal metabolism of 4-toluidines.

The influence of ortho chlorine substituents on 4-toluidine rat liver microsomal metabolism was investigated with 4-toluidine, 2-chloro-4-toluidine, and 2,6-dichloro-4-toluidine as substrates. Microsomal metabolic products were identified and quantified by HPLC, chemical assays, and synthesized reference compounds. Metabolites identified include products from aromatic ring hydroxylation, side-chain C-hydroxylation (benzyl alcohols and benzaldehydes), N-hydroxylation (hydroxylamines, nitrosotoluenes, azoxy, azo, and hydrazo derivatives), and two new types of microsomal metabolites: secondary amines [i.e. (halogenated) N-(4'-aminobenzyl)-4-toluidines] and an imine [i.e. N-(4'-amino-3',5'-dichlorobenzylidene)-2,6-dichloro-4-toluidine]. The secondary amine appeared to be a major microsomal metabolite for all three 4-toluidines studied. Quantification of the metabolite patterns demonstrated several influences of the chlorine substituents on metabolism of the 4-toluidine derivatives. The total conversion increased significantly in the order: 4-toluidine < 2-chloro-4-toluidine < 2,6-dichloro-4-toluidine, especially because of a marked increase in microsomal side-chain C-hydroxylation with increasing number of ortho chlorine substituents. The rate of N-hydroxylation varied much less. Aromatic ring hydroxylation was observed to a significant extent only for nonchlorinated 4-toluidine. Additional data from microsomal binding studies and molecular orbital calculations provided insight in possible mechanisms underlying the observed changes in metabolic profiles with increasing number of chlorine substituents at an ortho position with respect to the amine group.

Re-use of biologically treated wastewater of a brewery.

Despite excessive rainfalls, Flanders is dealing with a water deficiency. A rational use of water is a necessity. Apart from the prevention principle, the re-use of biologically treated wastewater (bio-effluent) is increasingly considered. From earlier research it is known that reverse osmosis (RO) is necessary for the elimination of salts and low molecular compounds from the bio-effluent. A thorough pretreatment is necessary to remove drastically the suspended solids, which are harmful to the reverse osmosis modules. This case study describes the experiments performed by SEGHERSbetter technology for Water N.V. on the treatment of wastewater effluent from a brewery. A comparative study between sand filtration (SF), in-line coagulation with sand filtration and ultrafiltration (UF) as a pretreatment was made. UF proved to be the best pretreatment for RO for the treatment of brewery bio-effluent. Finally, an economic evaluation of the membrane system (UF followed by RO) was made. The calculated total cost for the system is 0.26 Euros per m3 produced water. Investment costs and operation cost are also discussed.

Microperoxidase/H2O2-mediated alkoxylating dehalogenation of halophenol derivatives in alcoholic media.

The results of this study report the H2O2-driven microperoxidase-8 (MP8)-catalyzed dehalogenation of halophenols such as 4-fluorophenol, 4-chlorophenol, 4-bromophenol, and 2-fluorophenol in alcoholic solvents. In methanol, the conversion of the para-halophenols and 2-fluorophenol to, respectively, 4-methoxyphenol and 2-methoxyphenol, as the major dehalogenated products is observed. In ethanol, 4-ethoxyphenol is the principal dehalogenated product formed from 4-fluorophenol. Two mechanisms are suggested for this MP8-dependent alkoxylating dehalogenation reaction. In one of these mechanisms the oxene resonant form of compound I of MP8 is suggested to react with methanol forming a cofactor-peroxide-alkyl intermediate. This intermediate reacts with the reactive pi-electrons of the substrate, leading to the formation of the alkoxyphenols and the release of the fluorine substituent as fluoride anion.

The mercapturic acid biotransformation pathway of hexachlorobenzene is not involved in the induction of splenomegaly, or skin and lung lesions in the Brown Norway rat.

Involvement of the mercapturic acid pathway in the induction of splenomegaly and skin and lung pathology by hexachlorobenzene (HCB) in the rat was investigated by seeking to determine whether pentachloronitrobenzene (PCNB) has the same inflammatory effects as HCB, since both compounds are directly conjugated to glutathione, and further processed into the same mercapturic acid metabolites which are excreted via the urine. Female Brown Norway (BN/SsNO1aHsd) rats at 3 to 4 weeks of age were orally exposed to diets with or without supplementation with 450 mg HCB or equimolar (467 mg) or higher (934 mg) amounts of PCNB per kilogram of diet over 4 weeks. Gross skin lesion development and body weight gains were assessed during exposure and spleen and liver weights as well as histopathologic changes in skin and lung were assessed after exposure. After 3 weeks of exposure, urinary metabolites of the mercapturic acid and oxidative biotransformation pathways were identified using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Oral exposure of the rats to 450 mg/kg HCB resulted in an increase in relative spleen and liver weights as well as in the development of skin and lung pathology in the absence of overall liver toxicity. Equimolar or higher concentrations of PCNB caused none of these effects. Urinary levels of the mercapturic acid N-acetyl-S-(pentachlorophenyl)-cysteine (PCP-NAC), were comparable in HCB- and PCNB-treated rats. Levels of closely related methylsulfide derivatives of PCP-NAC, also generated via the same mercapturic acid pathway, appeared to be significantly higher in PCNB- than in HCB-treated rats, whereas the reverse was true for the urinary levels of the oxidative metabolite pentachlorophenol (PCP). Thus, results indicate that metabolites of the mercapturic acid pathway are not involved in the induction of splenomegaly and skin and lung pathology caused by HCB exposure in BN rats and that the main urinary metabolite of HCB in these BN rats is PCP. Since PCP itself, as well as other cytochrome P450-derived metabolites from HCB, are not likely to be involved in the induction of splenomegaly and skin and lung pathology, it is suggested that either the parent compound HCB or as-yet-unidentified non-P450-generated metabolites are involved in these inflammatory effects of HCB.

Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate.

Rat liver microsomal metabolism of 2-halogenated 4-methylanilines.

1. Rat liver microsomal metabolism of 2-fluoro-, 2-chloro- and 2-bromo-4-methylaniline was investigated using h.p.l.c. Metabolites identified include products from side-chain C-hydroxylation (benzyl alcohols and benzaldehydes) and N-hydroxylation (hydroxylamines and nitroso derivatives). Aromatic ring hydroxylation was not a major reaction pathway. 2. A new type of microsomal metabolite was detected which was identified as a secondary amine, i.e. a halogenated N-(4'-aminobenzyl)-4-methylaniline. 3. In addition to these products azoxy, azo and hydrazo derivatives were formed. 4. Benzyl alcohols and halogenated N-(4'-aminobenzyl)-4-methylanilines were the major microsomal metabolites for all three 2-halogenated 4-methylanilines. 5. Quantification of the metabolite patterns demonstrated an influence of the type of halogen substituent on the rate of microsomal metabolism. The rate of side-chain C-hydroxylation increases in the order 2-fluoro-4-methylaniline < 2-chloro-4-methylaniline < 2-bromo-4-methylaniline. 6. The rate of N-hydroxylation increases from 2-bromo-4-methylaniline < 2-fluoro-4-methylaniline < 2-chloro-4-methylaniline. That 2-chloro-4-methylaniline is N-hydroxylated to a larger extent is in accordance with its greater mutagenicity, twice that of 2-bromo-4-methylaniline.

Structure-activity study on the quinone/quinone methide chemistry of flavonoids.

A structure-activity study on the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids was performed. Using the glutathione trapping method followed by HPLC, (1)H NMR, MALDI-TOF, and LC/MS analysis to identify the glutathionyl adducts, the chemical behavior of the quinones/quinone methides of the different flavonoids could be deduced. The nature and type of mono- and diglutathionyl adducts formed from quercetin, taxifolin, luteolin, fisetin, and 3,3',4'-trihydroxyflavone show how several structural elements influence the quinone/quinone methide chemistry of flavonoids. In line with previous findings, glutathionyl adduct formation for quercetin occurs at positions C6 and C8 of the A ring, due to the involvement of quinone methide-type intermediates. Elimination of the possibilities for efficient quinone methide formation by (i) the absence of the C3-OH group (luteolin), (ii) the absence of the C2=C3 double bond (taxifolin), or (iii) the absence of the C5-OH group (3,3',4'-trihydroxyflavone) results in glutathionyl adduct formation at the B ring due to involvement of the o-quinone isomer of the oxidized flavonoid. The extent of di- versus monoglutathionyl adduct formation was shown to depend on the ease of oxidation of the monoadduct as compared to the parent flavonoid. Finally, unexpected results obtained with fisetin provide new insight into the quinone/quinone methide chemistry of flavonoids. The regioselectivity and nature of the quinone adducts that formed appear to be dependent on pH. At pH values above the pK(a) for quinone protonation, glutathionyl adduct formation proceeds at the A or B ring following expected quinone/quinone methide isomerization patterns. However, decreasing the pH below this pK(a) results in a competing pathway in which glutathionyl adduct formation occurs in the C ring of the flavonoid, which is preceded by protonation of the quinone and accompanied by H(2)O adduct formation, also in the C ring of the flavonoid. All together, the data presented in this study confirm that quinone/quinone methide chemistry can be far from straightforward, but the study provides significant new data revealing an important pH dependence for the chemical behavior of this important class of electrophiles.