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Douglas H Ohlendorf - One of the best experts on this subject based on the ideXlab platform.
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Encyclopedia of Inorganic and Bioinorganic Chemistry - Protocatechuate 3,4-Dioxygenase
Encyclopedia of Inorganic and Bioinorganic Chemistry, 2011Co-Authors: Douglas H Ohlendorf, M W VettingAbstract:A key step in the degradation of aromatic compounds in the biosphere is the ring-opening step. Intradiol Dioxygenases typically use a nonheme ferric iron to activate the substrate for an electrophilic attack by molecular oxygen to cleave catechol derivatives between the vicinal hydroxyls. Protocatechuate 3,4-dioxygenase (PCD) has been the most thoroughly studied of the intradiol Dioxygenases because of the presence of optical and electron paramagnetic resonance (EPR) spectroscopic signals. The structures of PCD from Pseudomonas putida and Acinetobacter calcoaceticus alone and in complexes with more than a dozen substrates and inhibitors have been used to visualize steps in substrate binding and ligand dissociation. 3D Structure Keywords: iron; non-Heme Proteins; mononuclear Iron Proteins; enzyme; protocatechuate 3; 4-dioxygenase; EC 1.13.11.13; non-heme iron oxidoreductase
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protocatechuate 3 4 dioxygenase
Encyclopedia of Inorganic and Bioinorganic Chemistry, 2006Co-Authors: Douglas H Ohlendorf, M W VettingAbstract:A key step in the degradation of aromatic compounds in the biosphere is the ring-opening step. Intradiol Dioxygenases typically use a nonheme ferric iron to activate the substrate for an electrophilic attack by molecular oxygen to cleave catechol derivatives between the vicinal hydroxyls. Protocatechuate 3,4-dioxygenase (PCD) has been the most thoroughly studied of the intradiol Dioxygenases because of the presence of optical and electron paramagnetic resonance (EPR) spectroscopic signals. The structures of PCD from Pseudomonas putida and Acinetobacter calcoaceticus alone and in complexes with more than a dozen substrates and inhibitors have been used to visualize steps in substrate binding and ligand dissociation. 3D Structure Keywords: iron; non-Heme Proteins; mononuclear Iron Proteins; enzyme; protocatechuate 3; 4-dioxygenase; EC 1.13.11.13; non-heme iron oxidoreductase
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Structure of catechol 1,2-dioxygenase from Pseudomonas arvilla.
Biochemical and Biophysical Research Communications, 2005Co-Authors: Cathleen A Earhart, M W Vetting, Lawrence Que, Ramachandraiah Gosu, Isabelle Michaud-soret, Douglas H OhlendorfAbstract:Catechol 1,2-dioxygenase was first studied by Hayaishi and colleagues in 1950. In 1967, catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 (PaCTD) was chosen as a model system for the catecholic intradiol Dioxygenases due to its activity, stability and expression level. Here we report the 2.65 A structure of the betabeta isozyme of PaCTD. The structure supports the hypothesis first made by Vetting and Ohlendorf [The 1.8A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker, Struct. Fold. Des. 8 (2000) 429-440.] that the catechol 1,2-Dioxygenases are lipid binding proteins. The 5 amino-terminal helices involved in dimerization and forming the lipid binding site are shown to be plastic in their positions and orientations. The sequence differences between the alpha and beta polypeptides are located at the part of the monomers distant from dimerization surface and thus permit the formation of the 3 isozymes (alphaalpha, alphabeta, and betabeta) of PaCTD. The reported inactivation by sulfhydryl-modifying reagents is explained by the structure. The 10-residue Helix F (residues 203-212) is proposed to be central in communicating between the lipid binding site and the active site.
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structure of catechol 1 2 dioxygenase from pseudomonas arvilla
Biochemical and Biophysical Research Communications, 2005Co-Authors: Cathleen A Earhart, M W Vetting, Lawrence Que, Ramachandraiah Gosu, Isabelle Michaudsoret, Douglas H OhlendorfAbstract:Abstract Catechol 1,2-dioxygenase was first studied by Hayaishi and colleagues in 1950. In 1967, catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 (PaCTD) was chosen as a model system for the catecholic intradiol Dioxygenases due to its activity, stability and expression level. Here we report the 2.65 A structure of the ββ isozyme of PaCTD. The structure supports the hypothesis first made by Vetting and Ohlendorf [The 1.8 A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker, Struct. Fold. Des. 8 (2000) 429–440.] that the catechol 1,2-Dioxygenases are lipid binding proteins. The 5 amino-terminal helices involved in dimerization and forming the lipid binding site are shown to be plastic in their positions and orientations. The sequence differences between the α and β polypeptides are located at the part of the monomers distant from dimerization surface and thus permit the formation of the 3 isozymes (αα, αβ, and ββ) of PaCTD. The reported inactivation by sulfhydryl-modifying reagents is explained by the structure. The 10-residue Helix F (residues 203–212) is proposed to be central in communicating between the lipid binding site and the active site.
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biophysical analyses of designed and selected mutants of protocatechuate 3 4 dioxygenase
Annual Review of Microbiology, 2004Co-Authors: Kent C Brown, M W Vetting, Cathleen A Earhart, Douglas H OhlendorfAbstract:▪ Abstract The catechol Dioxygenases allow a wide variety of bacteria to use aromatic compounds as carbon sources by catalyzing the key ring-opening step. These enzymes use specifically either catechol or protocatechuate (2,3-dihydroxybenozate) as their substrates; they use a bare metal ion as the sole cofactor. To learn how this family of metalloenzymes functions, a structural analysis of designed and selected mutants of these enzymes has been undertaken. Here we review the results of this analysis on the nonheme ferric iron intradiol dioxygenase protocatechuate 3,4-dioxygenase.
Jim C. Spain - One of the best experts on this subject based on the ideXlab platform.
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pathway and evolutionary implications of diphenylamine biodegradation by burkholderia sp strain js667
Applied and Environmental Microbiology, 2009Co-Authors: Kwanghee A Shin, Jim C. SpainAbstract:Diphenylamine (DPA) is a common contaminant at munitions-contaminated sites as well as at aniline manufacturing sites. Little is known about the biodegradation of the compound, and bacteria able to use DPA as the growth substrate have not been reported. Burkholderia sp. strain JS667 and Ralstonia sp. strain JS668 were isolated by selective enrichment from DPA-contaminated sediment. The isolates grew aerobically with DPA as the sole carbon, nitrogen, and energy source. During induction of DPA degradation, stoichiometric amounts of aniline accumulated and then disappeared, which suggested that aniline is on the DPA degradation pathway. Genes encoding the enzymes that catalyze the initial steps in DPA degradation were cloned from the genomic DNA of strain JS667. The Escherichia coli clone catalyzed stoichiometric transformation of DPA to aniline and catechol. Transposon mutagenesis, the sequence similarity of putative open reading frames to those of well-characterized Dioxygenases, and (18)O(2) experiments support the conclusion that the initial reaction in DPA degradation is catalyzed by a multicomponent ring-hydroxylating dioxygenase. DPA is converted to aniline and catechol via dioxygenation at the 1,2 position of the aromatic ring and spontaneous rearomatization. Aniline and catechol are further biodegraded by the well-established aniline degradation pathway. Genes that encode the complete aniline degradation pathway were found 12 kb downstream of the genes that encode the initial dioxygenase. Expression of the relevant Dioxygenases was confirmed by reverse transcription-PCR analysis. Both the sequence similarity and the gene organization suggest that the DPA degradation pathway evolved recently by the recruitment of two gene clusters that encode the DPA dioxygenase and aniline degradation pathway.
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Genetic and biochemical comparison of 2-aminophenol 1,6-dioxygenase of Pseudomonas pseudoalcaligenes JS45 to meta-cleavage Dioxygenases: divergent evolution of 2-aminophenol meta-cleavage pathway.
Archives of microbiology, 1999Co-Authors: John K. Davis, Charles C. Somerville, Jim C. SpainAbstract:Nitrobenzene is degraded to pyruvate and acetaldehyde by Pseudomonas pseudoalcaligenes JS45 via a reductive pathway, and by Comamonas sp. JS765 via an oxidative pathway. Although the initial reactions in the degradation of nitrobenzene by the two bacteria are totally different, the lower pathways are similar and converge at the level of 4-oxalocrotonate. In order to further investigate the biochemical properties and reveal the evolutionary relationships between the two lower pathways, the genes encoding the 2-aminophenol 1,6-dioxygenase were cloned and sequenced. 2-Aminophenol 1,6-dioxygenase from P. pseudoalcaligenes JS45 and catechol 2,3-dioxygenase from Comamonas sp. JS765 were able to act on both catechol and 2-aminophenol, but catechol was a suicide substrate of 2-aminophenol 1,6-dioxygenase. The activity of 2-aminophenol 1,6-dioxygenase was restored after removal of catechol and incubation with ascorbate and FeCl(2). Both the alpha-subunit (AmnA) and the beta-subunit (AmnB) of the dioxygenase from P. pseudoalcaligenes JS45 show a high degree of identity to the corresponding subunits of the ring-fission dioxygenase from Pseudomonas sp. AP-3: 67% for the alpha-subunit, and 84% for the beta-subunit. Sequence similarity studies suggest that the beta-subunits of both 2-aminophenol 1,6-Dioxygenases are distantly related to homoprotocatechuate 2,3-dioxygenase from Escherichia coli strains W and C and then to catechol 2, 3-dioxygenase from Alcaligenes eutrophus. Four active-site-relevant histidines are conserved in AmnB, but not in AmnA. The lack of conserved histidines indicates the absence of an Fe(2+) binding site in AmnA, which explains the previous observations of only approximately one Fe(2+) per two subunits in the 2-aminophenol 1, 6-Dioxygenases from P. pseudoalcaligenes JS45. The 2-aminophenol 1, 6-dioxygenase genes are located upstream of the 2-aminomuconic semialdehyde dehydrogenase gene, and a putative member of the YjgF protein family is upstream of the dioxygenase genes. Transcriptional analysis indicates that the YjgF-like protein, 2-aminophenol 1, 6-dioxygenase, and 2-aminomuconic semialdehyde dehydrogenase are coordinately transcribed. A putative ORF similar to part of the RNA helicase genes is downstream of the dehydrogenase gene. Both the novel organization of the genes and the phylogeny of the Dioxygenases and dehydrogenase indicate that the 2-aminophenol pathway in P. pseudoalcaligenes JS45 represents an example of a distant divergent evolution of meta-cleavage pathways.
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2 4 dinitrotoluene dioxygenase from burkholderia sp strain dnt similarity to naphthalene dioxygenase
Journal of Bacteriology, 1996Co-Authors: Wenchen Suen, B E Haigler, Jim C. SpainAbstract:2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by Dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.
Danuta Wojcieszynska - One of the best experts on this subject based on the ideXlab platform.
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Influence of metal ions on bioremediation activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2
World Journal of Microbiology and Biotechnology, 2013Co-Authors: Urszula Guzik, Katarzyna Hupert-kocurek, Karina Sałek, Danuta WojcieszynskaAbstract:The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different Dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe^3+, Cu^2+, Zn^2+, Co^2+, Al^3+, Cd^2+, Ni^2+ and Mn^2+ ions caused 20–80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the exception of Ni^2+) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three Dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas.
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characterization of catechol 2 3 dioxygenase from planococcus sp strain s5 induced by high phenol concentration
Acta Biochimica Polonica, 2012Co-Authors: Katarzyna Hupertkocurek, Urszula Guzik, Danuta WojcieszynskaAbstract:This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-Dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.
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high activity catechol 2 3 dioxygenase from the cresols degrading stenotrophomonas maltophilia strain kb2
International Biodeterioration & Biodegradation, 2011Co-Authors: Danuta Wojcieszynska, Katarzyna Hupertkocurek, Izabela Gren, Urszula GuzikAbstract:Abstract This study aimed at characterization of catechol 2,3-dioxygenase from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. 2-methylphenol, 3-methylphenol, and 4-methylphenol was completely degraded during 24 h in concentration 6 mM, 7 mM, and 5 mM, respectively. When cells of strain KB2 were growing on methylphenols, catechol 2,3-dioxygenase was induced. Biochemical analysis revealed that the examined enzyme was similar to another catechol 2,3-Dioxygenases, but showed extremely high activity. The enzyme was optimally active at 30 °C and pH 7.6. Kinetic studies showed that the value of K m , V max and Hill constant was 85.11 μM, 3.08 μM min −1 and 4.09 respectively. Comparative structural and phylogenetic analysis of catechol 2,3-dioxygenase from S. maltophilia KB2 had placed the protein with the single-ring substrate subfamily of the extradiol dioxygenase. We observed the presence of externally located α-helices and internally located β-sheets. We also suggest that the Fe 2+ ion binding is facilitated via four ligands: two histidine residues, one glutamate residue and one molecule of water.
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catechol 1 2 dioxygenase from the new aromatic compounds degrading pseudomonas putida strain n6
International Biodeterioration & Biodegradation, 2011Co-Authors: Urszula Guzik, Izabela Gren, Katarzyna Hupertkocurek, Danuta WojcieszynskaAbstract:Abstract This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida . When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K m and V max was 85.19 μM and 14.54 μM min −1 respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with cat A genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol Dioxygenases.
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induction of aromatic ring cleavage Dioxygenases in stenotrophomonas maltophilia strain kb2 in cometabolic systems
World Journal of Microbiology & Biotechnology, 2011Co-Authors: Danuta Wojcieszynska, Izabela Gren, Urszula Guzik, Magdalena Perkosz, Katarzyna HupertkocurekAbstract:Stenotrophomonas maltophilia KB2 is known to produce different enzymes of dioxygenase family. The aim of our studies was to determine activity of these enzymes after induction by benzoic acids in cometabolic systems with nitrophenols. We have shown that under cometabolic conditions KB2 strain degraded 0.25–0.4 mM of nitrophenols after 14 days of incubation. Simultaneously degradation of 3 mM of growth substrate during 1–3 days was observed depending on substrate as well as cometabolite used. From cometabolic systems with nitrophenols as cometabolites and 3,4-dihydroxybenzoate as a growth substrate, Dioxygenases with the highest activity of protocatechuate 3,4-dioxygenase were isolated. Activity of catechol 1,2- dioxygenase and protocatechuate 4,5-dioxygenase was not observed. Catechol 2,3-dioxygenase was active only in cultures with 4-nitrophenol. Ability of KB2 strain to induce and synthesize various Dioxygenases depending on substrate present in medium makes this strain useful in bioremediation of sites contaminated with different aromatic compounds.
Kenji Aoki - One of the best experts on this subject based on the ideXlab platform.
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Novel genes encoding 2-aminophenol 1,6-dioxygenase from Pseudomonas species AP-3 growing on 2-aminophenol and catalytic properties of the purified enzyme.
The Journal of biological chemistry, 1997Co-Authors: Shinji Takenaka, Shuichiro Murakami, Ryu Shinke, Kazuhisa Hatakeyama, Hideaki Yukawa, Kenji AokiAbstract:Abstract 2-Aminophenol 1,6-dioxygenase was purified from the cell extracts of Pseudomonas sp. AP-3 grown on 2-aminophenol. The product from 2-aminophenol by catalysis of the purified enzyme was identified as 2-aminomuconic 6-semialdehyde by gas chromatographic and mass spectrometric analyses. The molecular mass of the native enzyme was 140 kDa based on gel filtration. It was dissociated into molecular mass subunits of 32 (α-subunit) and 40 kDa (β-subunit) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase was a heterotetramer of α2β2. The genes coding for the α- and β-subunits of the enzyme were cloned and sequenced. Open reading frames of the genes (amnA and amnB) were 816 and 918 base pairs in length, respectively. The amino acid sequences predicted from the open reading frames of amnA andamnB corresponded to the NH2-terminal amino acid sequences of the α-subunit (AmnA) and β-subunit (AmnB), respectively. The deduced amino acid sequences of AmnB showed identities to some extent with HpaD (25.4%) and HpcB (24.4%) that are homoprotocatechuate 2,3-Dioxygenases from Escherichia coliW and C, respectively, belonging to class III in the extradiol Dioxygenases. On the other hand, AmnA had identity (23.3%) with only AmnB among the enzymes examined.
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Novel Genes Encoding 2-Aminophenol 1,6-Dioxygenase fromPseudomonas Species AP-3 Growing on 2-Aminophenol and Catalytic Properties of the Purified Enzyme
The Journal of biological chemistry, 1997Co-Authors: Shinji Takenaka, Shuichiro Murakami, Ryu Shinke, Kazuhisa Hatakeyama, Hideaki Yukawa, Kenji AokiAbstract:Abstract 2-Aminophenol 1,6-dioxygenase was purified from the cell extracts of Pseudomonas sp. AP-3 grown on 2-aminophenol. The product from 2-aminophenol by catalysis of the purified enzyme was identified as 2-aminomuconic 6-semialdehyde by gas chromatographic and mass spectrometric analyses. The molecular mass of the native enzyme was 140 kDa based on gel filtration. It was dissociated into molecular mass subunits of 32 (α-subunit) and 40 kDa (β-subunit) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase was a heterotetramer of α2β2. The genes coding for the α- and β-subunits of the enzyme were cloned and sequenced. Open reading frames of the genes (amnA and amnB) were 816 and 918 base pairs in length, respectively. The amino acid sequences predicted from the open reading frames of amnA andamnB corresponded to the NH2-terminal amino acid sequences of the α-subunit (AmnA) and β-subunit (AmnB), respectively. The deduced amino acid sequences of AmnB showed identities to some extent with HpaD (25.4%) and HpcB (24.4%) that are homoprotocatechuate 2,3-Dioxygenases from Escherichia coliW and C, respectively, belonging to class III in the extradiol Dioxygenases. On the other hand, AmnA had identity (23.3%) with only AmnB among the enzymes examined.
Urszula Guzik - One of the best experts on this subject based on the ideXlab platform.
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ORIGINAL PAPER Influence of metal ions on bioremediation activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas
2016Co-Authors: Urszula Guzik, Katarzyna Hupert-kocurek, Karina SałekAbstract:The Author(s) 2012. This article is published with open access at Springerlink.com Abstract The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different Dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe3?, Cu2?, Zn2?, Co2?, Al3?, Cd2?, Ni2? and Mn2? ions caused 20–80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the excep-tion of Ni2?) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three Dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas
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Influence of metal ions on bioremediation activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2
World Journal of Microbiology and Biotechnology, 2013Co-Authors: Urszula Guzik, Katarzyna Hupert-kocurek, Karina Sałek, Danuta WojcieszynskaAbstract:The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different Dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe^3+, Cu^2+, Zn^2+, Co^2+, Al^3+, Cd^2+, Ni^2+ and Mn^2+ ions caused 20–80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the exception of Ni^2+) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three Dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas.
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characterization of catechol 2 3 dioxygenase from planococcus sp strain s5 induced by high phenol concentration
Acta Biochimica Polonica, 2012Co-Authors: Katarzyna Hupertkocurek, Urszula Guzik, Danuta WojcieszynskaAbstract:This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-Dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.
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high activity catechol 2 3 dioxygenase from the cresols degrading stenotrophomonas maltophilia strain kb2
International Biodeterioration & Biodegradation, 2011Co-Authors: Danuta Wojcieszynska, Katarzyna Hupertkocurek, Izabela Gren, Urszula GuzikAbstract:Abstract This study aimed at characterization of catechol 2,3-dioxygenase from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. 2-methylphenol, 3-methylphenol, and 4-methylphenol was completely degraded during 24 h in concentration 6 mM, 7 mM, and 5 mM, respectively. When cells of strain KB2 were growing on methylphenols, catechol 2,3-dioxygenase was induced. Biochemical analysis revealed that the examined enzyme was similar to another catechol 2,3-Dioxygenases, but showed extremely high activity. The enzyme was optimally active at 30 °C and pH 7.6. Kinetic studies showed that the value of K m , V max and Hill constant was 85.11 μM, 3.08 μM min −1 and 4.09 respectively. Comparative structural and phylogenetic analysis of catechol 2,3-dioxygenase from S. maltophilia KB2 had placed the protein with the single-ring substrate subfamily of the extradiol dioxygenase. We observed the presence of externally located α-helices and internally located β-sheets. We also suggest that the Fe 2+ ion binding is facilitated via four ligands: two histidine residues, one glutamate residue and one molecule of water.
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catechol 1 2 dioxygenase from the new aromatic compounds degrading pseudomonas putida strain n6
International Biodeterioration & Biodegradation, 2011Co-Authors: Urszula Guzik, Izabela Gren, Katarzyna Hupertkocurek, Danuta WojcieszynskaAbstract:Abstract This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida . When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K m and V max was 85.19 μM and 14.54 μM min −1 respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with cat A genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol Dioxygenases.
