Biotransformation of 2,7-dichloro- and 1,2,3,4-tetrachlorodibenzo-p-dioxin by Sphingomonas wittichii RW1

Aerobic biotransformation of the diaryl ethers 2,7-dichlorodibenzo-p-dioxin and 1,2,3,4-tetrachlorodibenzo-p-dioxin by the dibenzo-p-dioxin-utilizing strain Sphingomonas wittichii RW1, producing corresponding metabolites, was demonstrated for the first time. Our strain transformed 2,7-dichlorodibenzo-p-dioxin, yielding 4-chlorocatechol, and 1,2,3,4-tetrachlorodibenzo-p-dioxin, producing 3,4,5,6-tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol; all of these compounds were unequivocally identified by mass spectrometry both before and after N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization by comparison with authentic standards. Additional experiments showed that strain RW1 formed a second metabolite, 2-methoxy-3,4,5,6-tetrachlorophenol, from the original degradation product, 3,4,5,6-tetrachlorocatechol, by methylation of one of the two hydroxy substituents.     

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. Received 19 May 1999/ Accepted in revised form 11 August 1999     

3-苯氧基苯甲酸降解菌Sphingomonas sp. SC-1降解苯酚环境条件及其降解中间产物的研究

【目的】探究高效降解3-苯氧基苯甲酸(3-Phenoxybenzoic acid,3-PBA)的鞘氨醇单胞菌(Sphingomonas sp.) SC-1对苯酚的降解特性。【方法】采用HPLC测定微生物降解体系中苯酚残留量,考察环境条件对菌株SC-1降解苯酚的影响;分析不同培养时间苯酚降解体系混合样品的HPLC谱图,确定其降解中间产物。【结果】菌株SC-1能在基础盐培养基中以苯酚为唯一碳源和能源生长,在初始pH 7.0、30 °C条件下,24 h可完全降解100 mg/L苯酚;Cu2+、Ba2+、Mn2+等对其降解苯酚有不同程度的抑制作用;HPLC谱图分析,初步确定邻苯二酚是菌株SC-1降解苯酚的中间产物,且该菌株可在48 h内完全降解100 mg/L邻苯二酚。【结论】菌株SC-1对苯酚及邻苯二酚均有较强的降解能力,为完善3-PBA的降解途径及污染3-PBA或含酚废水或含酚农药残留的降解提供了数据参考。     

Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1

Sphingomonas yanoikuyae B1 is able to utilize toluene, m-xylene, p-xylene, biphenyl, naphthalene, phenanthrene, and anthracene as sole sources of carbon and energy for growth. A forty kilobase region of DNA containing most of the genes for the degradation of these aromatic compounds was previously cloned and sequenced. Insertional inactivation of bphC results in the inability of B1 to grow on both polycyclic and monocyclic compounds. Complementation experiments indicate that the metabolic block is actually due to a polar effect on the expression of bphA3, coding for a ferredoxin component of a dioxygenase. Lack of the ferredoxin results in a nonfunctional polycyclic aromatic hydrocarbon dioxygenase and a nonfunctional toluate dioxygenase indicating that the electron transfer components are capable of interacting with multiple oxygenase components. Insertional inactivation of a gene for a dioxygenase oxygenase component downstream of bphA3 had no apparent effect on growth besides a polar effect on nahD which is only needed for growth of B1 on naphthalene. Insertional inactivation of either xylE or xylG in the meta-cleavage operon results in a polar effect on bphB, the last gene in the operon. However, insertional inactivation of xylX at the beginning of this cluster of genes does not result in a polar effect suggesting that the genes for the meta-cleavage pathway, although colinear, are organized in at least two operons. These experiments confirm the biological role of several genes involved in metabolism of aromatic compounds by S. yanoikuyae B1 and demonstrate the interdependency of the metabolic pathways for polycyclic and monocyclic aromatic hydrocarbon degradation. Received 13 May 1999/ Accepted in revised form 05 July 1999     

Pi binding by the F1-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane

Pi binding by the F(1)-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane (E. coli F(1)) was examined by two methods: the centrifuge column procedure [Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899] and the Paulus pressure dialysis cell [Paulus, H. (1969) Anal. Biochem. 32, 91-100]. The latter is an equilibrium dialysis-type procedure. Pi binding by beef heart F(1) could be determined by either procedure. However, direct binding of Pi to E. coli F(1) could be determined adequately only in the Paulus cell which indicated more than two binding sites per mol of enzyme with a K(d) in the range of 0.1 mM. It is concluded that previous failure to observe Pi binding to E. coli F(1) with the centrifuge column procedure is due to a rapid rate of dissociation of Pi from the E. coli enzyme which results in loss of Pi during transit of the enzyme-Pi complex through the column.     

Degradation of 4-chloro-2-methylphenol by an activated sludge isolate and its taxonomic description

The Gram-negative strain S1, isolated from activated sludge, metabolized 4-chloro-2-methylphenol by an inducible pathway via a modifiedortho-cleavage route as indicated by a transiently secreted intermediate, identified as 2-methyl-4-carboxymethylenebut-2-en-4-olide by gas chromatography/mass spectrometry. Beside 4-chloro-2-methylphenol only 2,4-dichlorophenol and 4-chlorophenol were totally degraded, without an accumulation of intermediates. The chlorinated phenols tested induced activities of 2,4-dichlorophenol hydroxylase and catechol 1,2-dioxygenase type II. Phenol itself appeared to be degraded more efficiently via a separate, inducibleortho-cleavage pathway. The strain was characterized with respect to its physiological and chemotaxonomic properties. The fatty acid profile, the presence of spermidine as main polyamine, and of ubiquinone Q-10 allowed the allocation of the strain into the -2 subclass of theProteobacteria. Ochrobactrum anthropi was indicated by fatty acid analysis as the most similar organism, however, differences in a number of physiological features (e.g. absence of nitrate reduction) and pattern of soluble proteins distinguished strain S1 from this species.     

Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport

The binding of radioactively labelled atrazin, metribuzin and phenmedipham by broken chloroplasts was studied. From the double-reciprocal plots (bound vs. free inhibitors) a high affinity binding reaction is graphically isolated which is related to the inhibition of photosynthetic electron transport. It is concluded that the specific binding sites correspond to the electron carrier molecules which are attacked by the inhibitors. The relative concentration of specific binding sites is 1 per 300–500 chlorophyll molecules.The binding of the labelled substances is competitively inhibited by each of the indicated unlabelled substances, by DCMU and by several pyridazinone derivatives. These results suggest that triazines, triazinones, pyridazinones, biscarbamates and phenylureas interfere with the same electron carrier of the photosynthetic electron transport chain, according to the same molecular mechanism.     

Preferential attack of the (S)-configured ether-linked carbons in bis-(1-chloro-2-propyl) ether by Rhodococcus sp. strain DTB

Rhodococcus sp. strain DTB (DSM 44534) was grown on a mixture of (R,R)-, (S,S)- and meso-bis-(1-chloro-2-propyl) ether (BCPE) as the sole source of carbon and energy. During BCPE degradation 1'-chloro-2'-propyl-3-chloro-2-prop-1-enyl-ether (DVE), 1-chloro-2-propanol and chloroacetone intermediates were formed. The BCPE or DVE stereoisomers were metabolized in consecutive order via scission of the ether bond, with discrimination against the (R) configuration. Resting cell suspensions of Rhodococcus pregrown on BCPE showed a preferential attack of the (S)-configured ether-linked carbons, resulting in an enantioselective enrichment of (R,R)-BCPE. Microbial discrimination of BCPE or DVE isomers and chemical conversion of the intermediates to 1-chloro-2-propanol allowed the identification of the configuration of all BCPE isomers and the DVE enantiomers. Elucidation of the absolute configuration of the 1-chloro-2-propanol isomers was achieved by enantioselective chemical synthesis.     

Selective and reversible inhibition of the ATPase of Micrococcus denitrificans by 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole

The covalent inhibitor of the beef heart mitochondrial ATPase 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole inhibits the ATPase of phosphorylating particles prepared from Micrococcus denitrificans. Inhibition of both ATP synthesis and ATP hydrolysis occurs at similar rates, with a similar pH dependence, and in each case the inhibition is relieved by treatment with dithiothreitol. These results are compared with those previously obtained with the mitochondrial ATPase.     

Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d

The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB , was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase ( ahdA ) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30–33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and ¹H-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.     

Catabolism of 4-hydroxy-2-trans-nonenal by THP1 monocytes/macrophages and inactivation of carboxylesterases by this lipid electrophile

Oxidative stress in cells and tissues leads to the formation of an assortment of lipid electrophiles, such as the quantitatively important 4-hydroxy-2-trans-nonenal (HNE). Although this cytotoxic aldehyde is atherogenic the mechanisms involved are unclear. We hypothesize that elevated HNE levels can directly inactivate esterase and lipase activities in macrophages via protein adduction, thus generating a biochemical lesion that accelerates foam cell formation and subsequent atherosclerosis. In the present study we examined the effects of HNE treatment on esterase and lipase activities in human THP1 monocytes/macrophages at various physiological scales (i.e., pure recombinant enzymes, cell lysate, and intact living cells). The hydrolytic activities of bacterial and human carboxylesterase enzymes (pnbCE and CES1, respectively) were inactivated by HNE in vitro in a time- and concentration-dependent manner. In addition, so were the hydrolytic activities of THP1 cell lysates and intact THP1 monocytes and macrophages. A single lysine residue (Lys105) in recombinant CES1 was modified by HNE via a Michael addition reaction, whereas the lone reduced cysteine residue (Cys389) was found unmodified. The lipolytic activity of cell lysates and intact cells was more sensitive to the inhibitory effects of HNE than the esterolytic activity. Moreover, immunoblotting analysis using HNE antibodies confirmed that several cellular proteins were adducted by HNE following treatment of intact THP1 monocytes, albeit at relatively high HNE concentrations (>50 μM). Unexpectedly, in contrast to CES1, the treatment of a recombinant human CES2 with HNE enhanced its enzymatic activity ∼3-fold compared to untreated enzyme. In addition, THP1 monocytes/macrophages can efficiently metabolize HNE, and glutathione conjugation of HNE is responsible for ∼43% of its catabolism. The functional importance of HNE-mediated inactivation of cellular hydrolytic enzymes with respect to atherogenesis remains obscure, although this study has taken a first step toward addressing this important issue by examining the potential of HNE to inhibit this biochemical activity in a human monocyte/macrophage cell line.     

Effects of 4-nitro- and 4-sulpho-7-substituted benzofurazans on nucleic acid and protein synthesis of a malignant fibrosarcoma cell line in combination with mild hyperthermia

The inhibitory effects of substituted nitro- and sulphobenzofurazans on DNA, RNA and protein synthesis were compared in a new malignant fibrosarcoma cell line at 37°C and 41°C. The effects of these drugs with and without mild hyperthermia were evaluated by determining the % inhibition of incorporation of ³H-precursors into DNA, RNA and protein. None of the sulphobenzofurazan derivatives (Sbf) were effective inhibitors of nucleic acid and protein synthesis at 37°C nor did they enhance the inhibitory effect of hyperthermia alone. The nitrobenzofurazan derivatives (Nbf) at concentrations 10% that used for the Sbf derivatives strongly inhibited biopolymer synthesis in a dose related manner; 4-chloro-7-nitrobenzofurazan (Nbf-Cl) being the most potent inhibitor. Hyperthermia amplified the effect of all the Nbf compounds tested on RNA and protein synthesis but did not further affect DNA synthesis. This selective synergistic effect was most pronounced when the lowest concentrations of Nbf compounds were studied. The synergism however, did not follow a uniform pattern. 6-Mercaptopurine and 6-(1-methyl-4-nitro-5-imidazoyl)thiopurine (Azathioprine) (100 μM) had marginal effects on nucleic acid and protein synthesis when the cells were exposed to these two thiopurines for 1 h at both 37°C and 41°C and they had only a moderate inhibitory effect after exposure for 15 h.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Metabolism of 4-chlorophenol by Azotobacter sp. GP1: Structure of the meta cleavage product of 4-chlorocatechol

Abstract A mutant strain of Azobacter sp. GP1 converted 4-chlorphenol to 4-chlorocatechol under cometabolic conditions. Under the same conditions the wild-type strain accumulated yellow compound, which by chemical and spectroscopic methods was identified as 5-chloro-2-hydroxy-6-oxohexadienoic acid (5-chloro-2-hydroxy-muconic semialdehyde). The structure of this compound indicates a meta -proximal cleavage of 4-chlorocatechol.     

Theoretical research in structure characteristics of different inhibitors and differences of binding modes with CBP bromodomain

The CBP (CREB (cAMP responsive element binding protein) binding protein) bromodomain (BRD) could recognize and bind with acetyl K382 of human tumor suppressor protein p53 which the mutation of encoding gene might cause human cancers. CBP-BRD serves as a promising drug target for several disease pathways and a series of effective drug have been discovered. In this study, molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GB/SA) approaches were performed to investigate the different binding modes between five inhibitors with CBP-BRD. Based on the energy and conformation analyses, a potent core fragment is chosen to act as the starting point for new inhibitor design by means of LUDI and rational drug design approaches. Then, T.E.S.T and molinspirition were applied to evaluate oral bioavailability and drug promiscuity of the new molecules. These results shed light on the idea for further inhibitor design.     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Isolation and characterization of a meta-cleavage product in the degradation of quinaldic acid by Azotobacter sp.

Abstract From different samples of soil seventeen strains were isolated which grew aerobically in mineral salts medium with quinaldic acid as sole carbon source. Mutants were induced with N -methyl- N '-nitro- N -nitrosoguanidine. One mutant could be isolated which accumulated a yellow compound. The properties of this purified compound were those expected for 2-oxo-3-(4'-hydroxy-2'-oxo-3',4'-en-butyrate)-pyridine-6-carboxylic acid.