Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran inSphingomonas sp. strain RW1

Bünz, Patricia V.; Falchetto, Rocco; Cook, Alasdair M.

doi:10.1007/bf00695119

Cited by 40 publications

(30 citation statements)

References 29 publications

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“…Finally, our phylogenetic analysis indicates that DxnB2 is most similar to hydrolases such as DxnB and CarC, which are involved in the degradation of dibenzofuran and the structurally related carbazole, respectively. The occurrence of multiple DxnB isozymes in S. wittichii RW1 is consistent with the proposal that this bacterium's ability to degrade a range of dioxins and dibenzofurans is due to the higher number of homologues of various aromatic catabolic genes present in its genome than in those of other bacterial strains (2)(3)(4).…”

Section: Discussionsupporting

confidence: 86%

“…For example, Armengaud and coworkers isolated four genes that encode ␣ subunits of ring-hydroxylating dioxygenases and two genes that encode ␤ subunits (2). The bacterium also contains at least two extradiol dioxygenases and three MCP hydrolases (2,4). Two of the MCP hydrolases, DxnB and DxnB2 (previously H1 and H2), were purified and shown to hydrolyze HOPDA and 8-hydroxy HOPDA (4).…”

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confidence: 99%

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Characterization of a C—C Bond Hydrolase fromSphingomonas wittichiiRW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites

Seah

Denis

et al. 2007

J Bacteriol

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Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k cat /K m ‫؍‬ 1.2 ؋ 10 7 M ؊1 s ؊1 ) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k cat /K m ‫؍‬ 1.7 ؋ 10 6 M ؊1 s ؊1 ), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.Chlorinated aromatic compounds, such as polychlorinated biphenyls (PCBs) and dibenzofurans, are among the most widespread, toxic, and/or persistent environmental pollutants. The Bph and Dxn/Dbf pathways responsible for the aerobic bacterial catabolism of biphenyl and dibenzofuran, respectively, have been extensively studied, in part due to their potential for remediating environments contaminated with the polychlorinated compounds (2, 10). The pathways share three homologous enzymes ( Fig. 1): a multicomponent dioxygenase that catalyzes the initial step of ring hydroxylation, an extradiol dioxygenase that catalyzes oxygenolytic cleavage of the catecholic intermediate at the meta position to yield 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) or ortho-substituted HOPDA, and a metacleavage product (MCP) hydrolase that catalyzes an unusual COC bond cleavage of the HOPDA.A general limitation of bacterial catabolic pathways for the degradation of complex industrial mixtures is that one or more pathway enzymes lack the requisite broad substrate specificity to degrade all man-made (xenobiotic) congeners of the naturally occurring compounds,...

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Section: Discussionsupporting

confidence: 86%

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confidence: 99%

Characterization of a C—C Bond Hydrolase fromSphingomonas wittichiiRW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites

Seah

Denis

et al. 2007

J Bacteriol

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“…strain DPO360, two different extradiol dioxygenases showing activity for 3-phenylcatechol and 2,2Ј,3-trihydroxybiphenyl and one C23O were detected in Sphingomonas sp. strain RW1 growing on dibenzofuran or salicylate (8). In dibenzofuran-grown Sphingomonas sp.…”

Section: Discussionmentioning

confidence: 99%

“…strain RW1 (9) as a four-component class IIA dioxygenase system. Ring cleavage of 2,2Ј,3-trihydroxybiphenyl was shown to be catalyzed by extradiol dioxygenases yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid (16, 59), which is subsequently hydrolyzed to 2-oxopent-4-enoate and salicylate (8,16,59). From Sphingomonas sp.…”

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confidence: 99%

Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360

et al. 1997

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The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K. Due to the environmental significance of its chlorinated derivatives, the metabolism of dibenzofuran has been studied intensively in the last few years (10, 14, 16-18, 48, 58, 59, 65). Accordingly, bacterial mineralization of dibenzofuran is initiated by dibenzofuran 4,4a-dioxygenase, catalyzing the angular dioxygenation of dibenzofuran. This gives rise to the chemically labile hemiacetal 4,4a-dihydro-4,4a-dihydroxydibenzofuran, which, after spontaneous rearomatization, yields 2,2Ј, 3-trihydroxybiphenyl (16, 59). A multicomponent dioxygenase of this angular dioxygenase type was purified from Sphingomonas sp. strain RW1 (9) as a four-component class IIA dioxygenase system. Ring cleavage of 2,2Ј,3-trihydroxybiphenyl was shown to be catalyzed by extradiol dioxygenases yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid (16, 59), which is subsequently hydrolyzed to 2-oxopent-4-enoate and salicylate (8,16,59). From Sphingomonas sp. strain RW1, a 2,2Ј,3-trihydroxybiphenyl-cleaving extradiol dioxygenase which was suggested to be involved in dibenzofuran mineralization was cloned and characterized (24). In this paper, three distinct extradiol dioxygenases from Terrabacter sp. strain DPO360 are shown to be involved in total mineralization of dibenzofuran and characterized according to their function in the degradative pathway.- MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. Terrabacter sp. strain DPO360 was isolated from tar-contaminated soil in Germany (57), and Terrabacter sp. strain DPO1361 was isolated from Rhine water (58). Both grampositive isolates were catalase positive and cytochrome oxidase negative. Biochemical characterization of these strains using the API-CH50 and API-20NE test kits (Biomerieux) gave identical results, apart from positive reactions for esculin and 5-ketogluconate with Terrabacter sp. strain DPO1361, reactions which were negative for Terrabacter sp. strain DPO360. Both strains were cultivated as described earlier (58) with dibenzofuran as a source of carbon. Yeast extract (0.005% [wt/vol]) was added to the minimal medium to supply vitamins. L broth (42) was used as a complex medium for Terrabacter sp. and Escherichia coli strains.[47] was used as a recipient for library construction and screening, and E. coli JM109 [recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ⌬(lac-proAB) FЈ(traD36 proAB ϩ lacI q lacZ⌬M15) [69] was used in all other cloning experiments. The cloning vectors pUC20 and pUC21 (identical with pUC20, except for an inverted polylinker) were obtained from Boehringer Mannheim Biochemicals. Plasmids constructed in the present study are shown in Fig. 3.Chemicals. Chemicals were of the highest purity commercially available (Merck, Darmstadt, Germany; Sigma-Aldrich, Steinheim, Germany). 3-Phenylcatechol was obtained from Wako Chemicals (Neuss, Germany). 3-C...

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“…However, characterization of catabolic meta-cleavage pathways of different aromatic compounds in the last years has led to the identification of an increasing number of hydrolases catalyzing C-C bond cleavage in different gram-positive and gram-negative bacterial species. Some of them have been purified and characterized (3,5,8,10,13,18,25,32).…”

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Identification of a Serine Hydrolase Which Cleaves the Alicyclic Ring of Tetralin

et al. 2000

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A gene designated thnD, which is required for biodegradation of the organic solvent tetralin by Sphingomonas macrogoltabidus strain TFA, has been identified. Sequence comparison analysis indicated that thnD codes for a carbon-carbon bond serine hydrolase showing highest similarity to hydrolases involved in biodegradation of biphenyl. An insertion mutant defective in ThnD accumulates the ring fission product which results from the extradiol cleavage of the aromatic ring of dihydroxytetralin. The gene product has been purified and characterized. ThnD is an octameric thermostable enzyme with an optimum reaction temperature at 65°C. ThnD efficiently hydrolyzes the ring fission intermediate of the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the ring fission product of the biphenyl meta-cleavage pathway. However, it is not active towards the equivalent intermediates of meta-cleavage pathways of monoaromatic compounds which have small substituents in C-6. When ThnD hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C bond comprised within the alicyclic ring of tetralin instead of cleaving a linear C-C bond, as all other known hydrolases of meta-cleavage pathways do. The significance of this activity of ThnD for the requirement of other activities to mineralize tetralin is discussed.

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Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran inSphingomonas sp. strain RW1

Cited by 40 publications

References 29 publications

Characterization of a C—C Bond Hydrolase fromSphingomonas wittichiiRW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites

Characterization of a C—C Bond Hydrolase fromSphingomonas wittichiiRW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites

Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360

Identification of a Serine Hydrolase Which Cleaves the Alicyclic Ring of Tetralin

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