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L Xun

Publications and source records attributed to L Xun.

31 records · Page 2Linked to original sources
PubMed1995

Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1.

Although reductive dehalogenation by anaerobic microorganisms offers great potential for the degradation of halocarbons, little is known about the biochemical mechanisms involved. It has previously been demonstrated that the dehalogenase activity involved in 3-chlorobenzoate dehalogenation by Desulfomonile tiedjei DCB-1 is present in the membrane fraction of the cell extracts. We report herein the purification of a 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of D. tiedjei DCB-1. The dehalogenase activity was monitored by the conversion of 3-chlorobenzoate to benzoate with reduced methyl viologen as a reducing agent. The membrane fraction of the cell extracts was obtained by ultracentrifugation, and the membrane proteins were solubilized with either the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) or Triton X-100 in the presence of glycerol. The solubilized dehalogenase was purified by ammonium sulfate fractionation and a combination of anion exchange, hydroxyapatite, and hydrophobic interaction chromatographies. This procedure yielded about 7% of the total dehalogenase activity with a 120-fold increase in specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified dehalogenase consisted of two subunits with molecular weights of 64,000 and 37,000. The enzyme converted 3-chlorobenzoate to benzoate at its highest specific activity in 10 mM potassium phosphate buffer (pH 7.2) at 38 degrees C. The enzyme was yellow and probably a heme protein. The enzyme had an adsorbance peak at 408 nm. The dithionite-reduced enzyme displayed absorbance peaks at 416, 522, and 550 nm. The dithionite-reduced enzyme was able to complex with carbon monoxide. The nature of the heme chromophore is currently unknown.

Benzoates↗
PubMed1994

Nucleotide sequence and functional analysis of the genes encoding 2,4,5-trichlorophenoxyacetic acid oxygenase in Pseudomonas cepacia AC1100.

Pseudomonas cepacia AC1100 is able to use the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. One of the early steps in this pathway is the conversion of 2,4,5-T to 2,4,5-trichlorophenol (2,4,5-TCP). 2,4,5-TCP accumulates in the culture medium when AC1100 is grown in the presence of 2,4,5-T. A DNA region from the AC1100 genome has been subcloned as a 2.7-kb SstI-XbaI DNA fragment, which on transfer to Pseudomonas aeruginosa PAO1 allows the conversion of 2,4,5-T to 2,4,5-TCP. We have determined the directions of transcription of these genes as well as the complete nucleotide sequences of the genes and the number and sizes of the polypeptides synthesized by pulse-labeling experiments. This 2.7-kb DNA fragment encodes two polypeptides with calculated molecular masses of 51 and 18 kDa. Proteins of similar sizes were seen in the T7 pulse-labeling experiment in Escherichia coli. We have designated the genes for these proteins tftA1 (which encodes the 51-kDa protein) and tftA2 (which encodes the 18-kDa protein). TftA1 and TftA2 have strong amino acid sequence homology to BenA and BenB from the benzoate 1,2-dioxygenase system of Acinetobacter calcoaceticus, as well as to XylX and XylY from the toluate 1,2-dioxygenase system of Pseudomonas putida. The Pseudomonas aeruginosa PAO1 strain containing the 2.7-kb SstI-XbaI fragment was able to convert not only 2,4,5-T to 2,4,5-TCP but also 2,4-dichlorophenoxyacetic acid to 2,4-dichlorophenol and phenoxyacetate to phenol.

Amino Acid Sequence↗
PubMed1993

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

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.

Amino Acid Sequence↗
PubMed1993

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

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.

Amino Acid Sequence↗
PubMed1992

Glutathione is the reducing agent for the reductive dehalogenation of tetrachloro-p-hydroquinone by extracts from a Flavobacterium sp.

Tetrachloro-p-hydroquinone is the first intermediate during pentachlorophenol degradation by Flavobacterium sp. strain ATCC 39723, a strict aerobe. We report here that tetrachlorohydroquinone was reductively dehalogenated to 2,3,6-trichloro-p-hydroquinone and subsequently to 2,6-dichloro-p-hydroquinone under anaerobic conditions by the cell extract from Flavobacterium. The reducing agent was identified to be the reduced form of glutathione. This is the first time glutathione has been identified as the reducing agent for reductive dehalogenation.

Anaerobiosis↗
PubMed1992

Confirmation of oxidative dehalogenation of pentachlorophenol by a Flavobacterium pentachlorophenol hydroxylase.

Pentachlorophenol (PCP) hydroxylase purified from Flavobacterium sp. strain ATCC 39723 converted PCP or 2,3,5,6-tetrachlorophenol to tetrachloro-p-hydroquinone (TeCH) with the co-consumption of O2 and NADPH. The purified enzyme incorporated 18O from 18O2 but not from H218O into the reaction end product TeCH. The results clearly demonstrate that PCP is oxidatively converted to TeCH by a monooxygenase-type enzyme from Flavobacterium sp. strain ATCC 39723.

Flavobacterium↗
PubMed1992

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

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.

Amino Acid Sequence↗
PubMed1992

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

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.

Aminophenols↗
PubMed1991

Biodegradation of triiodophenol by cell-free extracts of a pentachlorophenol-degrading Flavobacterium sp.

Pentachlorophenol (PCP) degrading Flavobacterium sp. ATCC 39723 was found to degrade other polyhalogenated phenolic compounds, including triiodophenol, tribromophenol, and trichlorophenol. Each compound was able to induce the degradation of the other compounds. A PCP Flavobacterium sp. mutant, F-2, was unable to degrade any of the halogenated compounds. The results suggest that all of the polyhalogenated phenols were degraded by the same enzyme system. This observation led us to exploit the sensitive leuco crystal violet assay, which measures the iodide released from triiodophenol. Cell free extracts from PCP-induced cells were able to release iodide from triiodophenol. The reaction required NADPH and oxygen.

Biodegradation, Environmental↗
PubMed1991

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

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.

Chromatography, Affinity↗
PubMed1988

Control of the Life Cycle of Methanosarcina mazei S-6 by Manipulation of Growth Conditions.

The morphology of Methanosarcina mazei was controlled by magnesium, calcium, and substrate concentrations and by inoculum size; these factors allowed manipulation of the morphology and interconversions between pseudosarcinal aggregates and individual, coccoid cells. M. mazei grew as aggregates in medium with a low concentration of catabolic substrate (either 50 mM acetate, 50 mM methanol, or 10 mM trimethylamine) unless Ca and Mg concentrations were high. Growth in medium high in Ca, Mg, and substrate (i.e., 150 mM acetate, 150 mM methanol, or 40 mM trimethylamine) converted pseudosarcinal aggregates to individual cocci. In such media, aggregates separated into individual cells which continued to grow exclusively as single cells during subsequent transfers. Conversion of single cells back to aggregates was complicated, because conditions which supported the aggregated morphology (e.g., low calcium or magnesium concentration) caused lysis of coccoid inocula. We recovered aggregates from coccoid cells by inoculating serial dilutions into medium high in calcium and magnesium. Cells from very dilute inocula grew into aggregates which disaggregated on continued incubation. However, timely transfer of the aggregates to medium low in calcium, magnesium, and catabolic substrates allowed continued growth as aggregates. We demonstrated the activity of the enzyme (disaggregatase) which caused the dispersion of aggregates into individual cells; disaggregatase was produced not only during disaggregation but also in growing cultures of single cells. Uronic acids, the monomeric constituents of the Methanosarcina matrix, were also produced during disaggregation and during growth as coccoids.

Journal Article↗
PubMed1987

Effects of pH, Temperature, and Nutrients on Propionate Degradation by a Methanogenic Enrichment Culture.

Enrichment cultures were used to determine the conditions promoting fastest methanogenic propionate degradation and growth by adapting the cultures to various physical and chemical conditions and measuring the specific growth rate. We found that the fastest growth of propionate oxidizers occurred at pH 6.8 to 8.5 and 32 to 45 degrees C. Acetate-degrading populations showed narrower optima for fastest growth (pH 6.8 to 7.2 and 37 to 43 degrees C). Enrichment cultures grew as well in minimal medium as in complex medium, although individual microbial populations appeared to require growth factors which could be met by cross-feeding.

Journal Article↗