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Danuta Wojcieszynska - One of the best experts on this subject based on the ideXlab platform.
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Research Article Degradation Potential of Protocatechuate 3,4-Dioxygenase from Crude Extract of Stenotrophomonas maltophilia Strain KB2 Immobilized in Calcium Alginate
2016Co-Authors: On Glyoxyl Agarose, Marta Krysiak, Urszula Guzik, Katarzyna Hupert-kocurek, Danuta WojcieszynskaAbstract:Copyright © 2014 Urszula Guzik et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microbial intradiol Dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties.This is the first report onuse of glyoxyl agarose and calciumalginate asmatrixes for the immobilization of protocatechuate 3,4-Dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of Dioxygenase in calciumalginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5∘Cand 10∘C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pHwhile immobilization of the enzyme on glyoxyl agarose did not influence pHprofile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calciumalginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions. 1
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degradation potential of protocatechuate 3 4 Dioxygenase from crude extract of stenotrophomonas maltophilia strain kb2 immobilized in calcium alginate hydrogels and on glyoxyl agarose
BioMed Research International, 2014Co-Authors: Urszula Guzik, Katarzyna Hupertkocurek, Marta Krysiak, Danuta WojcieszynskaAbstract:Microbial intradiol Dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-Dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of Dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5°C and 10°C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.
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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.
Urszula Guzik - One of the best experts on this subject based on the ideXlab platform.
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Research Article Degradation Potential of Protocatechuate 3,4-Dioxygenase from Crude Extract of Stenotrophomonas maltophilia Strain KB2 Immobilized in Calcium Alginate
2016Co-Authors: On Glyoxyl Agarose, Marta Krysiak, Urszula Guzik, Katarzyna Hupert-kocurek, Danuta WojcieszynskaAbstract:Copyright © 2014 Urszula Guzik et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microbial intradiol Dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties.This is the first report onuse of glyoxyl agarose and calciumalginate asmatrixes for the immobilization of protocatechuate 3,4-Dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of Dioxygenase in calciumalginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5∘Cand 10∘C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pHwhile immobilization of the enzyme on glyoxyl agarose did not influence pHprofile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calciumalginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions. 1
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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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degradation potential of protocatechuate 3 4 Dioxygenase from crude extract of stenotrophomonas maltophilia strain kb2 immobilized in calcium alginate hydrogels and on glyoxyl agarose
BioMed Research International, 2014Co-Authors: Urszula Guzik, Katarzyna Hupertkocurek, Marta Krysiak, Danuta WojcieszynskaAbstract:Microbial intradiol Dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-Dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of Dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5°C and 10°C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.
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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.
Timothy D. H. Bugg - One of the best experts on this subject based on the ideXlab platform.
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enzymology of the carotenoid cleavage Dioxygenases reaction mechanisms inhibition and biochemical roles
Archives of Biochemistry and Biophysics, 2014Co-Authors: Peter J Harrison, Timothy D. H. BuggAbstract:Carotenoid cleavage Dioxygenases (CCDs) are a large family of non-heme iron (II) dependent enzymes. CCDs catalyse the selective oxidative cleavage of carotenoids to produce apocarotenoids. Apocarotenoid derived molecules form important signalling molecules in plants in the form of abscisic acid and strigolactone and in mammals in the form of retinal. Very little is known biochemically about the CCDs and only a handful of CCDs have been biochemically characterised. Mechanistically, debate surrounds whether CCDs utilise a mono or Dioxygenase mechanism. Here, we review the biochemical roles of CCDs, discuss the mechanisms by which CCD cleavage is proposed to occur, and discuss recent reports of selective CCD enzyme inhibitors.
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acid base catalysis in the extradiol catechol Dioxygenase reaction mechanism site directed mutagenesis of his 115 and his 179 in escherichia coli 2 3 dihydroxyphenylpropionate 1 2 Dioxygenase mhpb
Biochemistry, 2004Co-Authors: Sharon Mendel, And Andrew Arndt, Timothy D. H. BuggAbstract:The extradiol catechol Dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid−base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol Dioxygenases, positioned within 4−5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-Dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid−base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half...
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cis trans isomerization of a cyclopropyl radical trap catalyzed by extradiol catechol Dioxygenases evidence for a semiquinone intermediate
Journal of the American Chemical Society, 1996Co-Authors: Emma L Spence, And John G Langley, Timothy D. H. BuggAbstract:Substrate analogues cis- and trans-2-(2,3-dihydroxyphenyl)cyclopropane-1-carboxylic acid were synthesized as probes for a semiquinone radical intermediate in the (2,3-dihydroxyphenyl)propionate 1,2-Dioxygenase reaction. These analogues were found to be substrates for oxidative cleavage by extradiol Dioxygenases from Escherichia coli and Alcaligenes eutrophus. The stereochemistry of the ring fission products was analyzed by conversion to cyclopropane-1,2-dicarboxylic acids using the ensuing hydrolase enzyme MhpC, followed by GCMS analysis. This analysis revealed 85−94% trans product and 6−15% cis products, implying that cis/trans isomerization of the cyclopropyl ring substituents had taken place during the enzymatic conversion. These results are consistent with a reversible opening of the cyclopropyl ring, and hence consistent with the intermediacy of a semiquinone radical intermediate in the extradiol catechol Dioxygenase reaction.
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catechol Dioxygenases from escherichia coli mhpb and alcaligenes eutrophus mpci sequence analysis and biochemical properties of a third family of extradiol Dioxygenases
Journal of Bacteriology, 1996Co-Authors: E L Spence, M Kawamukai, J Sanvoisin, H Braven, Timothy D. H. BuggAbstract:The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-Dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol Dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-Dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-Dioxygenase from E. coli, and with human 3-hydroxyanthranilate Dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of Dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol Dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.
Douglas H Ohlendorf - One of the best experts on this subject based on the ideXlab platform.
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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 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.
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the 1 8 a crystal structure of catechol 1 2 Dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker
Structure, 2000Co-Authors: M W Vetting, Douglas H OhlendorfAbstract:Abstract Background: Intradiol Dioxygenases catalyze the critical ring-cleavage step in the conversion of catecholate derivatives to citric acid cycle intermediates. Catechol 1,2-Dioxygenases (1,2-CTDs) have a rudimentary design structure — a homodimer with one catalytic non-heme ferric ion per monomer, that is (αFe 3+ ) 2 . This is in contrast to the archetypical intradiol Dioxygenase protocatechuate 3,4-Dioxygenase (3,4-PCD), which forms more diverse oligomers, such as (αβFe 3+ ) 2–12 . Results : The crystal structure of 1,2-CTD from Acinetobacter sp. ADP1 (Ac 1,2-CTD) was solved by single isomorphous replacement and refined to 2.0 A resolution. The structures of the enzyme complexed with catechol and 4-methylcatechol were also determined at resolutions of 1.9 A and 1.8 A, respectively. While the characteristics of the iron ligands are similar, Ac 1,2-CTD differs from 3,4-PCDs in that only one subunit is used to fashion each active-site cavity. In addition, a novel ‘helical zipper', consisting of five N-terminal helices from each subunit, forms the molecular dimer axis. Two phospholipids were unexpectedly found to bind within an 8 × 35 A hydrophobic tunnel along this axis. Conclusions: The helical zipper domain of Ac 1,2-CTD has no equivalent in other proteins of known structure. Sequence analysis suggests the domain is a common motif in all members of the 1,2-CTD family. Complexes with catechol and 4-methylcatechol are the highest resolution complex structures to date of an intradiol Dioxygenase. Furthermore, they confirm several observations seen in 3,4-PCDs, including ligand displacement upon binding exogenous ligands. The structures presented here are the first of a new family of intradiol Dioxygenases.
Gerben J Zylstra - One of the best experts on this subject based on the ideXlab platform.
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dna stable isotope probing integrated with metagenomics for retrieval of biphenyl Dioxygenase genes from polychlorinated biphenyl contaminated river sediment
Applied and Environmental Microbiology, 2009Co-Authors: John F Quensen, Laurie Seliger, Tamara V Tsoi, Gerben J Zylstra, Joonhong Park, Jorge L M Rodrigues, James M TiedjeAbstract:Stable isotope probing with [ 13 C]biphenyl was used to explore the genetic properties of indigenous bacteria able to grow on biphenyl in PCB-contaminated River Raisin sediment. A bacterial 16S rRNA gene clone library generated from [ 13 C]DNA after a 14-day incubation with [ 13 C]biphenyl revealed the dominant organisms to be members of the genera Achromobacter and Pseudomonas . A library built from PCR amplification of genes for aromatic-ring-hydroxylating Dioxygenases from the [ 13 C]DNA fraction revealed two sequence groups similar to bphA (encoding biphenyl Dioxygenase) of Comamonas testosteroni strain B-356 and of Rhodococcus sp. RHA1. A library of 1,568 cosmid clones was produced from the [ 13 C]DNA fraction. A 31.8-kb cosmid clone, detected by aromatic Dioxygenase primers, contained genes of biphenyl Dioxygenase subunits bphAE , while the rest of the clone9s sequence was similar to that of an unknown member of the Gammaproteobacteria . A discrepancy in G+C content near the bphAE genes implies their recent acquisition, possibly by horizontal transfer. The biphenyl Dioxygenase from the cosmid clone oxidized biphenyl and unsubstituted and para -only-substituted rings of polychlorinated biphenyl (PCB) congeners. A DNA-stable isotope probing-based cosmid library enabled the retrieval of functional genes from an uncultivated organism capable of PCB metabolism and suggest dispersed Dioxygenase gene organization in nature.
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microbial Dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation
Applied and Environmental Microbiology, 2006Co-Authors: Sini Ad Ni M Chadhain, Sean R Norman, Karen V Pesce, Jerome J Kukor, Gerben J ZylstraAbstract:The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH Dioxygenase enzymes. These Rieske primers were employed to track Dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference Dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the Dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel Dioxygenase genes.
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molecular and biochemical characterization of two meta cleavage Dioxygenases involved in biphenyl and m xylene degradation by beijerinckia sp strain b1
Journal of Bacteriology, 1995Co-Authors: Eungbin Kim, Gerben J ZylstraAbstract:Beijerinckia sp. strain B1 is able to grow on either biphenyl or m-xylene as the sole source of carbon and is capable of cooxidizing many polycyclic aromatic hydrocarbons. The catabolic pathways for biphenyl and m-xylene degradation are coinduced and share common downstream enzymatic reactions. The catabolic pathway for biphenyl degradation involves two meta-cleavage steps, one for 2,3-dihydroxybiphenyl and a second for catechol. The catabolic pathway for m-xylene involves one m-cleavage step for 3-methylcatechol. The genes for two meta-cleavage Dioxygenases were cloned from Beijerinckia sp. strain B1 on a single fragment of genomic DNA. The two genes are located approximately 5.5 kb away from one another. Expression of each gene separately in Escherichia coli and analysis of the meta-cleavage Dioxygenase produced showed that one enzyme was more specific for 2,3-dihydroxybiphenyl while the second was more specific for catechol. The genes for the two meta-cleavage enzymes were thus labeled bphC and xylE for 2,3-dihydroxybiphenyl 1,2-Dioxygenase and catechol 2,3-Dioxygenase, respectively. Nondenaturing polyacrylamide gel electrophoresis followed by enzyme activity staining showed that the two meta-cleavage Dioxygenases could be easily separated from each other. Similar analyses of Beijerinckia sp. strain B1 grown on succinate, biphenyl, or m-xylene indicate that both meta-cleavage enzymes are induced when cells are grown on either biphenyl or m-xylene. The nucleotide sequence was determined for both bphC and xylE. The two genes are transcribed in opposite directions, demonstrating that at least two operons must be involved in biphenyl degradation by Beijerinckia sp. strain B1. Analysis of the deduced amino acid sequence indicates that 2,3-dihydroxybiphenyl 1,2-Dioxygenase (BphC) falls into the class of meta-cleavage Dioxygenases acting on dihydroxylated polycyclic aromatic hydrocarbons and is somewhat distinct from the main group of meta-cleavage Dioxygenases acting on 2,3-dihydroxybiphenyl. Catechol 2,3-Dioxygenase (XyIE) falls into the class of meta-cleavage enzymes acting on dihydroxylated monocyclic aromatic hydrocarbons but shows little similarity to the canonical TOL plasmid-encoded catechol 2,3-Dioxygenase.