Degradation of 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1.

Pseudomonas sp. strain HBP1 was found to grow on 2-hydroxy- and 2,2'-dihydroxy-biphenyl as the sole carbon and energy sources. The first step in the degradation of these compounds was catalyzed by an NADH-dependent monooxygenase. The enzyme inserted a hydroxyl group adjacent to the already existing hydroxyl group to form 2,3-dihydroxybiphenyl when acting on 2-hydroxybiphenyl and to form 2,2',3-trihydroxybiphenyl when acting on 2,2'-dihydroxybiphenyl. To be substrates of the monooxygenase, compounds required a 2-hydroxyphenyl-R structure, with R being a hydrophobic group (e.g., methyl, ethyl, propyl, sec-butyl, phenyl, or 2-hydroxyphenyl). Several chlorinated hydroxybiphenyls served as pseudosubstrates by effecting consumption of NADH and oxygen without being hydroxylated. Further degradation of 2,3-dihydroxy- and 2,2',3-trihydroxybiphenyl involved meta cleavage, with subsequent formation of benzoate and salicylate, respectively.     

Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1.

A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.     

Biodegradation of Dibenzo-p-dioxin, Dibenzofuran, and Chlorodibenzo-p-dioxins by Pseudomonas veronii PH-03

The dioxin-degrading strain Pseudomonas veronii PH-03 was isolated from contaminated soil by selective enrichment techniques. Strain PH-03 grew on dibenzo-p-dioxin and dibenzofuran as a sole carbon source. Further, 1-chlorodibenzo-p-dioxin, 2-chlorodibenzo-p-dioxin and other dioxin metabolites, salicylic acid, and catechol were also metabolized well. Resting cells of strain PH-03 transformed dibenzo-p-dioxin, dibenzofuran, 2,2',3-trihydroxybiphenyl, and some chlorodioxins to their corresponding metabolic intermediates such as catechol, salicylic acid, 2-hydroxy-(2-hydroxyphenoxy)-6-oxo-2,4-hexadienoic acid, and chlorocatechols. The formation of these metabolites was confirmed by comparison of gas chromatography-mass spectrometry (GC-MS) data with those of authentic compounds. Although we did observe the production of 3,4,5,6-tetrachlorocatechol (3,4,5,6-TECC) from 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) with resting cell suspensions of PH-03, growth of strain PH-03 in the presence of 1,2,3,4-TCDD was poor. This result suggests that strain PH-03 is unable to utilize 3,4,5,6-TECC, even at very low concentration (0.01 mM) due to its toxicity. In cell-free extracts of DF-grown cells, 2,2',3-trihydroxybiphenyl dioxygenase, 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase, and catechol-2,3-dioxygense activities were detected. Moreover, the activities of meta-pyrocatechase and 2,2',3-trihydroxybiphenyl dioxygenase from the crude cell-free extracts were inhibited by 3-chlorocatechol. However, no inhibition was observed in intact cells when 3-chlorocatechol was formed as intermediate.     

Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1.

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing 2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation, hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain RW1 when this organism was grown with dibenzo-p-dioxin.     

Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1.

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing 2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation, hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain RW1 when this organism was grown with dibenzo-p-dioxin.     

Transformation of 3-chlorodibenzofuran by Pseudomonas sp. HH69

The dibenzofuran-degrading bacterial strain Pseudomonas sp. HH69 showed high oxidative activity towards 3-chlorodibenzofuran (3CDF). During the co-metabolic turnover of 3CDF large amounts of 4-chlorosalicylate and temporarily small amounts of salicylate were excreted. Simultaneously a yellow colour appeared due to the excretion of two polar products. Conversion of 3CDF by a mutant, derived from Pseudomonas sp. HH69 and defective in 2,3-dihydroxybiphenyl-1,2-dioxygenase led to the formation of equal quantities of 4'-chloro-2,2',3-trihydroxybiphenyl (4'CTHBP) and 4-chloro-2,2',3-trihydroxybiphenyl (4CTHBP). Crude extracts of the wild type transformed 4'CTHBP to 4-chlorosalicylate, whilst 4CTHBP was transformed to salicylate. Hence, we propose a non-selective initial attack on both aromatic rings of 3CDF and a degradative pathway for the resulting chlorotrihydroxybiphenyls.     

Bacterial degradation of aromatic compounds via angular dioxygenation

Dioxygenation is one of the important initial reactions of the bacterial degradation of various aromatic compounds. Aromatic compounds, such as biphenyl, toluene, and naphthalene, are dioxygenated at lateral positions of the aromatic ring resulting in the formation of cis-dihydrodiol. This "normal" type of dioxygenation is termed lateral dioxygenation. On the other hand, the analysis of the bacterial degradation of fluorene (FN) analogues, such as 9-fluorenone, dibenzofuran (DF), carbazole (CAR), and dibenzothiophene (DBT)-sulfone, and DF-related diaryl ether compounds, dibenzo-p-dioxin (DD) and diphenyl ether (DE), revealed the presence of the novel mode of dioxygenation reaction for aromatic nucleus, generally termed angular dioxygenation. In this atypical dioxygenation, the carbon bonded to the carbonyl group in 9-fluorenone or to heteroatoms in the other compounds, and the adjacent carbon in the aromatic ring are both oxidized. Angular dioxygenation of DF, CAR, DBT-sulfone, DD, and DE produces the chemically unstable hemiacetal-like intermediates, which are spontaneously converted to 2,2',3-trihydroxybiphenyl, 2'-aminobiphenyl-2,3-diol, 2',3'-dihydroxybiphenyl-2-sulfinate, 2,2',3-trihydroxydiphenyl ether, and phenol and catechol, respectively. Thus, angular dioxygenation for these compounds results in the cleavage of the three-ring structure or DE structure. The angular dioxygenation product of 9-fluorenone, 1-hydro-1,1a-dihydroxy-9-fluorenone is a chemically stable cis-diol, and is enzymatically transformed to 2'-carboxy-2,3-dihydroxybiphenyl. 2'-Substituted 2,3-dihydroxybiphenyls formed by angular dioxygenation of FN analogues are degraded to monocyclic aromatic compounds by meta cleavage and hydrolysis. Thus, after the novel angular dioxygenation, subsequent degradation pathways are homologous to the corresponding part of that of biphenyl. Compared to the bacterial strains capable of catalyzing lateral dioxygenation, few bacteria having angular dioxygenase have been reported. Only a few degradation pathways, CAR-degradation pathway of Pseudomonas resinovorans strain CA10, DF/DD-degradation pathway of Sphingomonas wittichii strain RW1, DF/DD/FN-degradation pathway of Terrabacter sp. strain DBF63, and carboxylated DE-degradation pathway of P. pseudoalcaligenes strain POB310, have been investigated at the gene level. As a result of the phylogenetic analysis and the comparison of substrate specificity of angular dioxygenase, it is suggested that this atypical mode of dioxygenation is one of the oxygenation reactions originating from the relaxed substrate specificity of the Rieske nonheme iron oxygenase superfamily. Genetic characterization of the degradation pathways of these compounds suggests the possibility that the respective genetic elements constituting the entire catabolic pathway have been recruited from various other bacteria and/or other genetic loci, and that these pathways have not evolutionary matured.     

Molecular bases of aerobic bacterial degradation of dioxins: involvement of angular dioxygenation

In the last decade, extensive investigation has been done on the bacterial degradation of dioxins and its related compounds, because this class of chemicals is highly toxic and has been widely distributed in the environment. These studies have revealed the primary importance of a novel dioxygenation reaction, called angular dioxygenation, in the aerobic bacterial degradation pathway of dioxin. Accompanied by the electron transport proteins, Rieske nonheme iron oxygenase catalyzes the incorporation of oxygen atoms to the ether bond-carrying carbon (the angular position) and an adjacent carbon, resulting in the irreversible cleavage of the recalcitrant aryl ether bond. The 2,2',3-trihydroxybiphenyl or 2,2',3-trihydroxydiphenyl ether derivatives formed are degraded through meta cleavage. In addition to the degradation system of dibenzofuran and dibenzo-p-dioxin (the nonchlorinated model compounds of dioxin), those of fluorene and carbazole were shown to function in dioxin degradation. Some dioxin degradation pathways have been studied biochemically and genetically. In addition, feasibility studies have shown that some dioxin-degrading strains can function in actual dioxin-contaminated soil. These studies provide useful information for the establishment of a bioremediation method for dioxin contamination. This review summarizes recent progress on molecular and biochemical bases of the bacterial aerobic degradation of dioxin and related compounds.     

Evolutionarily divergent extradiol dioxygenases possess higher specificities for polychlorinated biphenyl metabolites

The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorinated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp. strain LB400 (DHBDLB400), DHBDP6-I and DHBDP6-III from Rhodococcus globerulus P6, and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. strain RW1 (THBDRW1). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the Kmapp values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorinated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3',5'-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophobic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHBDP6-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHBDP6-III than was DHB. Indeed, DHBDP6-III had the highest apparent specificity for 4,3',5'-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THBDRW1 had the highest specificity for 2'-Cl DHB and was at least five times more resistant to inactivation by 2'-Cl DHB, consistent with the similarity between the latter and 2,2',3-trihydroxybiphenyl. Nonetheless, THBDRW1 had the lowest specificity for 2',6'-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (kcatapp < 0.001 s(-1)).     

Transformation of polychlorinated biphenyls by a novel BphA variant through the meta-cleavage pathway

The transformation of 20 polychlorinated biphenyls (PCBs) through the meta-cleavage pathway by recombinant Escherichia coli cells expressing the bphEFGBC locus from Burkholderia cepacia LB400 and the bphA genes from different sources was compared. The analysis of PCB congeners for which hydroxylation was observed but no formation of the corresponding yellow meta-cleavage product demonstrated that only lightly chlorinated congeners including one tetrachlorobiphenyl (2,2',4,4'-CB) were transformed into their corresponding yellow meta-cleavage products. Although many other tetrachlorobiphenyls (2, 2',5,5'-CB, 2,2',3,5'-CB, 2,4,4',5-CB, 2,3',4',5-CB, 2,3',4,4'-CB) and one pentachlorobiphenyl (2,2',4,5,5'-CB) tested were depleted from resting cell suspensions, no yellow meta-cleavage products were observed. For most of these congeners, dihydrodiol compounds accumulated as the endproducts, indicating that the bphB-encoded biphenyl-2,3-dihydrodiol-2,3-dehydrogenase is a key limiting step for further degradation of highly chlorinated congeners. These results suggest that engineering the biphenyl dioxygenase alone is insufficient for an improved removal of PCB. Rather, improved degradation of PCBs is more likely to be achieved with recombinant strains containing metabolic pathways not only specifically engineered for expanding the initial dioxygenation but also for the mineralization of PCBs.     

Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360.

The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K.-H. Engesser, V. Strubel, K. Christoglou, P. Fischer, and H. G. Rast, FEMS Microbiol. Lett. 65:205-210, 1989; V. Strubel, Ph.D. thesis, University of Stuttgart, Stuttgart, Germany, 1991; V. Strubel, H. G. Rast, W. Fietz, H.-J. Knackmuss, and K.-H. Engesser, FEMS Microbiol. Lett. 58:233-238, 1989). Two 2,3-dihydroxybiphenyl-1,2-dioxygenases (BphC1 and BphC2) and one catechol-2,3-dioxygenase (C23O) were shown to be expressed in Terrabacter sp. strain DPO360 growing with dibenzofuran as a sole source of carbon and energy. These enzymes exhibited strong sensitivity to oxygen. They were purified to apparent homogeneity as homodimers (BphC and BphC2) and as a homotetrameric catechol-2,3-dioxygenase (C23O). According to their specificity constants kcat/Km, both BphC1 and BphC2 were shown to be responsible for the cleavage of 2,2',3-trihydroxybiphenyl, the first metabolite in dibenzofuran mineralization along the angular dioxygenation pathway. With this substrate, BphC2 exhibited a considerably higher kcat/Km, value (183 microM/min) than BphC1 (29 microM/min). Catechol-2,3-dioxygenase was recognized to be not involved in the ring cleavage of 2,2',3-trihydroxybiphenyl (kcat/Km, 1 microM/min). Analysis of deduced amino acid sequence data of bphC1 revealed 36% sequence identity to nahC from Pseudomonas putida PpG7 (S. Harayama and M. Rekik, J. Biol. Chem. 264:15328-15333, 1989) and about 40% sequence identity to various bphC genes from different Pseudomonas and Rhodococcus strains. In addition, another 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (bphC3) was cloned from the genome of Terrabacter sp. strain DPO360. Expression of this gene, however, could not be detected in Terrabacter sp. strain DPO360 after growth with dibenzofuran.     

Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain.

Propylene-grown Xanthobacter cells (strain Py2) degraded several chlorinated alkenes of environmental concern, including trichloroethylene, 1-chloroethylene (vinyl chloride), cis- and trans-1,2-dichloroethylene, 1,3-dichloropropylene, and 2,3-dichloropropylene. 1,1-Dichloroethylene was not degraded efficiently, while tetrachloroethylene was not degraded. The role of alkene monooxygenase in catalyzing chlorinated alkene degradations was established by demonstrating that glucose-grown cells which lack alkene monooxygenase and propylene-grown cells in which alkene monooxygenase was selectively inactivated by propyne were unable to degrade the compounds. C2 and C3 chlorinated alkanes were not oxidized by alkene monooxygenase, but a number of these compounds were inhibitors of propylene and ethylene oxidation, suggesting that they compete for binding to the enzyme. A number of metabolites enhanced the rate of degradation of chlorinated alkenes, including propylene oxide, propionaldehyde, and glucose. Propylene stimulated chlorinated alkene oxidation slightly when present at a low concentration but became inhibitory at higher concentrations. Toxic effects associated with chlorinated alkene oxidations were determined by measuring the propylene oxidation and propylene oxide-dependent O2 uptake rates of cells previously incubated with chlorinated alkenes. Compounds which were substrates for alkene monooxygenase exhibited various levels of toxicity, with 1,1-dichloroethylene and trichloroethylene being the most potent inactivators of propylene oxidation and 1,3- and 2,3-dichloropropylene being the most potent inactivators of propylene oxide-dependent O2 uptake. No toxic effects were seen when cells were incubated with chlorinated alkenes anaerobically, indicating that the product(s) of chlorinated alkene oxidation mediates toxicity.     

Inhibitory effects of 2-substituted-1-naphthol derivatives on cyclooxygenase I and II

Naphthol derivatives, 2-(3'-hydroxypropyl)-naphthalen-1-ol (2), 2-(3'-hydroxy-2'-methylpropyl)-naphthalen-1-ol (3) and 2-(3'-hydroxy-2',2'-dimethylpropyl)-naphthalen-1-ol (7) were synthesized and already reported by our group. Therefore in this paper we described further synthesis of their ether derivatives, 3-(1-methoxy-naphthalen-2-yl)-propan-1-ol (4), 3-(1-methoxy-naphthalen-2-yl)-2methyl-propan-1-ol (5), 3-(1-methoxy-naphthalen-2-yl)-2,2-dimethyl-propan-1-ol (8), 2-(3-methoxy-propyl)-naphthalen-1-ol (10) and 2-(3-methoxy-2,2-dimethyl-propyl)-naphthalen-1-ol (13). Compounds 4, 5 and 8 were prepared by methylation of compounds 2, 3 and 7, respectively while compounds 10 and 13 were prepared in good yield from naphthols 2 and 7, respectively. When tested for inhibitory activity, five compounds (2, 3, 7, 10 and 13) showed preferential inhibition of COX-2 over COX-1, while compounds 4, 5 and 8 lacked inhibitory effect on either the COX-1 or COX-2 isozyme. The structure-activity relationships of these naphthols analyzed by docking experiments, indicated that the presence of hydroxyl group at C-1 position on the naphthalene nucleus enhanced the anti-inflammatory activity towards COX-2 via hydrogen bonding to the COX-2 Val 523 side chain. When this hydroxyl group was replaced by methoxy group, there was no inhibition. C-2' Dimethyl substituents on the propyl chain also increased the inhibitory activity. All active compounds have the C-1 hydroxyl group aligned so as to form hydrogen bond with Val 523. The results provide a model for the binding of the naphthol derivatives to COX-2 and facilitate the design of more potent or selective analogs prior to synthesis.     

Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain.

Propylene-grown Xanthobacter cells (strain Py2) degraded several chlorinated alkenes of environmental concern, including trichloroethylene, 1-chloroethylene (vinyl chloride), cis- and trans-1,2-dichloroethylene, 1,3-dichloropropylene, and 2,3-dichloropropylene. 1,1-Dichloroethylene was not degraded efficiently, while tetrachloroethylene was not degraded. The role of alkene monooxygenase in catalyzing chlorinated alkene degradations was established by demonstrating that glucose-grown cells which lack alkene monooxygenase and propylene-grown cells in which alkene monooxygenase was selectively inactivated by propyne were unable to degrade the compounds. C2 and C3 chlorinated alkanes were not oxidized by alkene monooxygenase, but a number of these compounds were inhibitors of propylene and ethylene oxidation, suggesting that they compete for binding to the enzyme. A number of metabolites enhanced the rate of degradation of chlorinated alkenes, including propylene oxide, propionaldehyde, and glucose. Propylene stimulated chlorinated alkene oxidation slightly when present at a low concentration but became inhibitory at higher concentrations. Toxic effects associated with chlorinated alkene oxidations were determined by measuring the propylene oxidation and propylene oxide-dependent O2 uptake rates of cells previously incubated with chlorinated alkenes. Compounds which were substrates for alkene monooxygenase exhibited various levels of toxicity, with 1,1-dichloroethylene and trichloroethylene being the most potent inactivators of propylene oxidation and 1,3- and 2,3-dichloropropylene being the most potent inactivators of propylene oxide-dependent O2 uptake. No toxic effects were seen when cells were incubated with chlorinated alkenes anaerobically, indicating that the product(s) of chlorinated alkene oxidation mediates toxicity.     

Dioxygenolytic cleavage of aryl ether bonds: 1, 10-dihydro-1, 10-dihydroxyfluoren-9-one, a novel arene dihydrodiol as evidence for angular dioxygenation of dibenzofuran

Two dibenzofuran degrading bacteria, Brevibacterium strain DPO 1361 and strain DPO 220, were found to utilize fluorene as sole source of carbon and energy. Cells which were grown on dibenzofuran, transformed fluorene into a number of products. For five of the seven metabolites isolated, the structure could be established unequivocally. Accumulation of one metabolite, 1,10-dihydroxy-1,10-dihydrofluoren-9-one, indicated the presence of a novel type of dioxygenase, attacking polynuclear aromatic systems in the unusual angular position. Debenzofuran degradation is proposed to likewise proceed via initial angular dioxygenation. One aryl oxygen ether bond, which normally is extremely stable, is thus transformed to a hemiacetal. After spontaneous cleavage and subsequent rearomatization by dehydration, 2,2',3-trihydroxybiphenyl [3-(2-hydroxyphenyl)-catechol] thus results as the immediate product of the first enzymatic reaction in the degradation sequence.     

7-deazainosine derivatives: synthesis and characterization of 7- and 7,8-substituted pyrrolo [2,3-d]pyrimidine ribonucleosides

The synthesis of model 7 deazapurine derivatives related to tubercidin and toyocamycin has been performed. Tubercidin derivatives were obtained by simple conversion of the amino group of the heterocyclic moiety of the starting 7-deazadenosine compounds, into a hydroxyl group. Preparation of toyocamycin derivatives was accomplished by treatment of the silylated 6-bromo-5-cyanopyrrolo[2,3-d]pyrimidin-4-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-d-ribofuranose. The glycosylation reaction afforded a mixture of 8-bromo 7-cyano 2',3',5' tri-O-benzoyl 7-deazainosine and 6-bromo-5-cyano-3-(2',3',5'-tri-O-benzoyl-beta-d-ribofuranosyl)pyrrolo[2,3-d]-pyrimidin-4-one isomers: The structures were assigned on the basis of NMR spectroscopy studies. Next deprotection treatment gave the novel 7-deazainosine ribonucleosides.     

Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl metabolites in isolated rat hepatocytes

Polychlorinated biphenyl (PCB) and PCB metabolites are highly lipophilic and accumulate easily in the lipid bilayer and fat deposits of the body. The molecular cytotoxic mechanisms of these metabolites are still not understood. The aim of the present study was to compare the cytotoxicity and toxicological properties of six dihydroxylated metabolites using isolated rat hepatocytes. All of the metabolites were more cytotoxic than 4-chlorobiphenyl (4-ClBP) and less cytotoxic than phenyl hydroquinone (PHQ). The order of cytotoxic effectiveness of catecholic metabolites expressed as LC(50) (2h) was 3',4'-diCl-2,3-diOH-biphenyl>PHQ>4'-Cl-2,5-diOH-biphenyl, 4'-Cl-2,3-diOH-biphenyl>2',5'-diCl-3,4-diOH-biphenyl>2',3'-diCl-3,4-diOH-biphenyl>3',4'-diCl-3,4-diOH-biphenyl>4'Cl-3,4-diOH-biphenyl>4'-Cl-biphenyl; showing that the positions of hydroxyl and chlorine groups were important for their hepatotoxicity and that the two 2,3-diOH congeners were the most cytotoxic. Cytotoxicity for 3,4-diOH metabolites correlated with the number and position of chlorine atoms with the more chlorine atoms being more cytotoxic. The cytotoxic order of metabolites with two chlorine atoms being 2',5'>2',3'>3',4'. Borneol, an uridine diphosphate glucuronosyltransferases (UGT) inhibitor, increased the cytotoxicity of all tested metabolites; suggesting that glucuronidation was a major mechanism of elimination of these compounds. On the other hand entacapone, a catechol-O-methyl transferase (COMT) inhibitor, only increased the cytotoxicity of 3',4'-diCl-3,4-diOH-biphenyl, 3',4'-diCl-2,3-diOH-biphenyl and 4'-Cl-2,3-diOH-biphenyl. Hepatocyte GSH was depleted (oxidized and conjugated) by these metabolites before cytotoxicity ensued in a similar order of effectiveness to their cytotoxicity with PHQ being the most effective. Hepatocyte mitochondrial membrane potential also decreased before cytotoxicity ensued with a similar order of effectiveness as their cytotoxicity. These results suggest that catecholic cytotoxicity can be attributed to mitochondrial toxicity and oxidative stress. Semiquinone or benzoquinone species were also important in the cytotoxicity of catecholic metabolites.     

Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L

Two antioxidant compounds were isolated from C. sappan L by multiple steps of column chromatography and thin layer chromatography in succession with superoxide scavenging assay as activity monitor. Structures of the two compounds were determined by spectroscopic methods as 1',4'-dihydro-spiro[benzofuran-3(2H),3'-[3H-2]benzopyran]-1',6',6',7'-tetrol (compound 1) and 3-[[4,5-dihydroxy-2(hydroxymethyl) phenyl]-methyl]-2,3-dihydro-3,6-benzofurandiol (compound 2). Characterization of antioxidant properties of these two compounds was done by determining the inhibitory effect on xanthine oxidase activity as well as scavenging effect on superoxide anion and hydroxyl radicals. Our results indicated that compounds 1 and 2 inhibited xanthine oxidase activity and scavenged superoxide anion and hydroxyl radicals. Compounds 1 and 2 possessed similar radical scavenging activities as ascorbic acid, and they were more effective than other well-known antioxidants such as alpha-tocopherol, beta-carotene, and BHT. As inhibitors of free radical formation, compounds 1 and 2 were more effective than all the other antioxidants tested. In conclusion, compounds 1 and 2 can be regarded as primary antioxidants with radical-scavenging and chain-breaking activities as well as secondary antioxidants with inhibitory effect on radical generation.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.