The glutathione S-transferases (GST) represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C, synthetase, are encoded by single genes and both have arisen separately from the soluble GST. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress.A description of the mechanisms of transcriptional and posttranscriptional regulat...

The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.

Nitroaromatic compounds are released into the biosphere almost exclusively from anthropogenic sources. Some compounds are produced by incomplete combustion of fossil fuels; others are used as synthetic intermediates, dyes, pesticides, and explosives. Recent research revealed a number of microbial systems capable of transforming or biodegrading nitroaromatic compounds. Anaerobic bacteria can reduce the nitro group via nitroso and hydroxylamino intermediates to the corresponding amines. Isolates of Desulfovibrio spp. can use nitroaromatic compounds as their source of nitrogen. They can also reduce 2,4,6-trinitrotoluene to 2,4,6-triaminotoluene. Several strains of Clostridium can catalyze a similar reduction and also seem to be able to degrade the molecule to small aliphatic acids. Anaerobic systems have been demonstrated to destroy munitions and pesticides in soil. Fungi can extensively degrade or mineralize a variety of nitroaromatic compounds. For example, Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene and 2,4,6-trinitrotoluene and shows promise as the basis for bioremediation strategies. The anaerobic bacteria and the fungi mentioned above mostly transform nitroaromatic compounds via fortuitous reactions. In contrast, a number of nitroaromatic compounds can serve as growth substrates for aerobic bacteria. Removal or productive metabolism of nitro groups can be accomplished by four different strategies. (a) Some bacteria can reduce the aromatic ring of dinitro and trinitro compounds by the addition of a hydride ion to form a hydride-Meisenheimer complex, which subsequently rearomatizes with the elimination of nitrite. (b) Monooxygenase enzymes can add a single oxygen atom and eliminate the nitro group from nitrophenols. (c) Dioxygenase enzymes can insert two hydroxyl groups into the aromatic ring and precipitate the spontaneous elimination of the nitro group from a variety of nitroaromatic compounds. (d) Reduction of the nitro group to the corresponding hydroxylamine is the initial reaction in the productive metabolism of nitrobenzene, 4-nitrotoluene, and 4-nitrobenzoate. The hydroxylamines undergo enzyme-catalyzed rearrangements to hydroxylated compounds that are substrates for ring-fission reactions. Potential applications of the above reactions include not only the biodegradation of environmental contaminants, but also biocatalysis and synthesis of valuable organic molecules.

Biodegradation of Nitroaromatic Compounds

Microbial reductive dehalogenation.

Structure and function of glutathione S-transferases

https://www.itqb.unl.pt/~imartins/Czaplicka_2004.pdf

Sources and transformations of chlorophenols in the natural environment.

Tetrachloro-p-hydroquinone (TeCH) is the first intermediate in pentachlorophenol (PCP) degradation by Flavobacterium sp strain ATCC 39723 We previously purified a PCP hydroxylase that oxidized PCP to TeCH Subsequently, we identified the reductive dehalogenation of TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone in a cell extract with the reduced form of glutathione as the reducing agent under anaerobic conditions Here we report the purification of a TeCH reductive dehalogenase that reductively dehalogenated TeCH to trichlorohydroquinone and then to dichlorohydroquinone The enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, and phenyl-agarose, anion-exchange, and gel filtration column chromatographies As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the protein has a molecular weight of about 30,000; nondenaturing polyacrylamide gel electrophoresis analysis suggests that the native enzyme exists as a dimer The enzyme used glutathione but not NADPH, NADH, dithiothreitol, or ascorbic acid as the reducing agent The optimal pH was close to neutral

Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp.

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light. Images

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

The gene pcpC, encoding tetrachloro-p-hydroquinone (TeCH) reductive dehalogenase, was cloned from Flavobacterium sp strain ATCC 39723 and sequenced The gene was identified by hybridization with a degenerate oligonucleotide designed from the N-terminal sequence of the purified protein An open reading frame of 747 nucleotides was found, which predicts a translational product of 248 amino acids having a molecular weight of 28,263, which agrees favorably with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-determined molecular weight of 30,000 reported for the purified protein The predicted translational product of pcpC matched the N-terminal sequence of the purified protein exactly From the nucleotide sequence, the protein appears to have a processed formylmethionyl An Escherichia coli pcpC overexpression clone was shown to produce dichlorohydroquinone and trichlorohydroquinone from TeCH Protein data base searches grouped the predicted translational sequence of pcpC with two previously reported plant glutathione S-transferases but less significantly with any of the mammalian glutathione S-transferases or the glutathione-utilizing, hydrolytic dechlorinating enzyme from Methylobacterium sp strain DM4

Characterization of a Flavobacterium glutathione S-transferase gene involved reductive dechlorination.

The pcpB gene of Flavobacterium sp. strain ATCC 39723 was cloned by using a degenerate primer designed from the N-terminal sequence of the purified enzyme. The nucleotide sequence of pcpB was determined and found to encode an open reading frame of 1,614 nucleotides, yielding a predicted translation product of 538 amino acids, in agreement with the estimated size of the purified protein analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The transcriptional start of pcpB was found to be 80 bp upstream of the translational start, and the transcript was found to be induced in Flavobacterium sp. strain ATCC 39723 by the presence of pentachlorophenol but to be constitutive in the Escherichia coli pcpB clone. DNA hybridizations with genomic DNAs from Arthrobacter sp. strain ATCC 33790 and Pseudomonas sp. strain SR3 revealed a similar-size 3.0-kb EcoRI fragment, whereas there was no positive hybridization with genomic DNA from Rhodococcus chlorophenolicus. Cell extracts from an E. coli pcpB overexpression strain, as well as the whole cells, were proficient in the dechlorination of pentachlorophenol to tetrachlorohydroquinone. Protein data base comparisons of the predicted translation products revealed regions of homology with other microbial monooxygenases, including phenol-2-monooxygenase and tryptophan-2-monooxygenase.

Cloning, sequence analysis, and expression of the Flavobacterium pentachlorophenol-4-monooxygenase gene in Escherichia coli.

An understanding of the enzymatic reactions catalyzing the degradation of substituted phenols, a major group of environmental pollutants, is required for the development of biological methods for the decontamination of halophenol-polluted sites. We found that a flavomonooxygenase, pentachlorophenol hydroxylase, isolated from a Flavobacterium sp., catalyzed a primary attack on a broad range of substituted phenols, hydroxylating the para position and removing halogen, nitro, amino, and cyano groups to produce halide, nitrite, hydroxylamine, and cyanide, respectively. Elimination of 1 mol of a halogen, nitro, or cyano group required 2 mol of NADPH, while only 1 mol of NADPH was required to remove 1 mol of an amino group or hydrogen.

Luying Xun

Papers

Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp.

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

Characterization of a Flavobacterium glutathione S-transferase gene involved reductive dechlorination.

Cloning, sequence analysis, and expression of the Flavobacterium pentachlorophenol-4-monooxygenase gene in Escherichia coli.

Diverse substrate range of a Flavobacterium pentachlorophenol hydroxylase and reaction stoichiometries.