benzofurans and 2-chlorodibenzofuran

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) is a Rieske-type oxygenase that catalyzes the stereospecific oxygenation of many heterocyclic aromatics including dibenzofuran. In a previous work, we evolved BphAE(LB400) and obtained BphAE(RR41). This variant metabolizes dibenzofuran and 2-chlorodibenzofuran more efficiently than BphAE(LB400). However, the regiospecificity of BphAE(RR41) toward these substrates differs. Dibenzofuran is metabolized principally through a lateral dioxygenation whereas 2-chlorodibenzofuran is metabolized principally through an angular dioxygenation. In order to explain this difference, we examined the crystal structures of both substrate-bound forms of BphAE(RR41) obtained under anaerobic conditions. This structure analysis, in combination with biochemical data for a Ser283Gly mutant provided evidences that the substrate is compelled to move after oxygen-binding in BphAE(RR41):dibenzofuran. In BphAE(RR41):2-chlorodibenzofuran, the chlorine atom is close to the side chain of Ser283. This contact is missing in the BphAE(RR41):dibenzofuran, and strong enough in the BphAE(RR41):2-chlorodibenzofuran to help prevent substrate movement during the catalytic reaction.

Topics: Benzofurans; Burkholderia; Catalysis; Crystallization; Dioxygenases; Glycine; Mutation; Protein Conformation; Serine

Chlorination and thermal degradation of 2-chlorodibenzodioxin and dibenzofuran were investigated using sealed tubes at 350 degrees C in the presence of CuCl(2) or CuCl. Twelve organic species, including polychlorobenzenes, were identified and quantified as a function of residence time. Time behaviours of PCDDs and PCDFs regarding destruction processes were compared. The formation of polychlorobenzenes from the thermal heterogeneous degradation of PCDDs was only a minor pathway but it could contribute to the good linear correlation observed between the concentration of polychlorobenzenes and those of PCDD/Fs in the emissions of municipal solid waste incinerators.

Topics: Benzofurans; Carbon Dioxide; Chlorobenzenes; Copper; Dibenzofurans, Polychlorinated; Dioxins; Halogenation; Hot Temperature; Polychlorinated Dibenzodioxins

Rhodococcus sp. strain HA01, isolated through its ability to utilize dibenzofuran (DBF) as the sole carbon and energy source, was also capable, albeit with low activity, of transforming dibenzo-p-dioxin (DD). This strain could also transform 3-chlorodibenzofuran (3CDBF), mainly by angular oxygenation at the ether bond-carrying carbon (the angular position) and an adjacent carbon atom, to 4-chlorosalicylate as the end product. Similarly, 2-chlorodibenzofuran (2CDBF) was transformed to 5-chlorosalicylate. However, lateral oxygenation at the 3,4-positions was also observed and yielded the novel product 2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran. Two gene clusters encoding enzymes for angular oxygenation (dfdA1A2A3A4 and dbfA1A2) were isolated, and expression of both was observed during growth on DBF. Heterologous expression revealed that both oxygenase systems catalyze angular oxygenation of DBF and DD but exhibited complementary substrate specificity with respect to CDBF transformation. While DfdA1A2A3A4 oxygenase, with high similarity to DfdA1A2A3A4 oxygenase from Terrabacter sp. strain YK3, transforms 3CDBF by angular dioxygenation at a rate of 29% +/- 4% that of DBF, 2CDBF was not transformed. In contrast, DbfA1A2 oxygenase, with high similarity to the DbfA1A2 oxygenase from Terrabacter sp. strain DBF63, exhibited complementary activity with angular oxygenase activity against 2CDBF but negligible activity against 3CDBF. Thus, Rhodococcus sp. strain HA01 constitutes the first described example of a bacterial strain where coexpression of two angular dioxygenases was observed. Such complementary activity allows for the efficient transformation of chlorinated DBFs.

Topics: Benzofurans; Dioxins; Dioxygenases; DNA, Bacterial; DNA, Ribosomal; Gene Expression Profiling; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Multigene Family; Oxidation-Reduction; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Rhodococcus; RNA, Ribosomal, 16S; Salicylates; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Substrate Specificity

Topics: Abnormalities, Drug-Induced; Animals; Benzofurans; Body Weight; Bone Development; Embryonic and Fetal Development; Female; Male; Mice; Mice, Inbred ICR; Organ Size; Pregnancy

When 2-chloro[14C]dibenzofuran was intravenously administered to the rats, about 86% of radioactivity was found in the urine, the large intestinal contents and the feces within 24 h. Approximately 3% of 2-chlorodibenzofuran (2CDF) radioactivity was present in the adipose tissue 48 h after an intravenous administration. A similar excretion pattern of the compound was observed in orally administered rats. From experiments in vitro, approximately 80% of 2CDF was present in the red blood cell fraction and the remainder in the plasma fraction. Bile cannulation studies revealed involvement of enterohepatic circulation in the metabolism of the compound. Bile specimens that were subjected to enzymatic hydrolysis by arylsulfatase and/or beta-glucuronidase showed the presence of various yet unidentified conjugated substances. A comparative study using 3-chlorodibenzofuran (3CDF) showed a similar distribution pattern in the 2CDF-treated rats, but 2CDF appeared to accumulate more in the adipose tissue and red blood cells. The present study demonstrates that most of 2CDF is quickly metabolized to give rise to a number of metabolites and excreted from the body, but suggests that the compound once distributed in the adipose tissue may remain there for a relatively long period of time.

Topics: Animals; Arylsulfatases; Benzofurans; Bile; Feces; Gastrointestinal Contents; Glucuronidase; Hydrolysis; Male; Rats; Rats, Inbred Strains; Tissue Distribution

The hepatic metabolism of 2-chlorodibenzofuran was investigated in the rat. When 2-chloro[14C]dibenzofuran was intravenously administered to bile duct-cannulated rats, about 90% of radioactivity was excreted in bile and urine within 6 hr, the bile being the preferential route. Another group of rats received oral administration of cold 2-chlorodibenzofuran and bile fluid was collected by surgical bile duct cannulation for qualitative analysis. Three hydroxylated metabolites were isolated by high performance liquid chromatography from the bile fluid after hydrolytic digestion with sulfatase and beta-glucuronidase and were identified by mass and nuclear magnetic resonance spectrometries to be 2-chloro-3,7-dihydroxydibenzofuran, 2-chloro-3-hydroxydibenzofuran and 2-chloro-7-hydroxydibenzofuran, respectively. Analyses of the radioactive bile fluid by thin layer chromatography revealed that there was no detectable amounts of unmetabolized 2-chlorodibenzofuran in the bile fluid and the hydroxylated metabolites were present not as aglycons but as conjugated substances. The results suggest that 2-chlorodibenzofuran is rapidly metabolized in the rat, and the 3 and/or 7 positions play an important role in the metabolism of 2-chlorodibenzofuran.

Topics: Animals; Benzofurans; Bile; Chromatography, High Pressure Liquid; Injections, Intravenous; Male; Rats; Rats, Inbred Strains