Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100

Daubaras, Dayna L.; Hershberger, C. Douglas; Kitano, Kazuaki; Chakrabarty, Ananda M.

doi:10.1128/aem.61.4.1279-1289.1995

Cited by 73 publications

(33 citation statements)

References 62 publications

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“…Even though the highest level of homology found is only 32%, amino acids highly conserved in the alpha, pi, mu, and theta classes of the GST superfamily (53) are present in the predicted amino acid sequence of ORF 3. GST-encoding genes, such as bphK (23), or ORF 3 in the tft operon (12), have been found in numerous operons or gene clusters specifying ring cleavage and further metabolism of aromatic compounds, thus suggesting a role of GST in these pathways. Crawford and Frick reported a glutathione-dependent isomerization of maleylpyruvate to fumarylpyruvate in the gentisate pathway in Moraxella (10).…”

Section: Resultsmentioning

confidence: 99%

“…Analysis of its deduced amino acid sequence revealed significant homology to glutathione reductases of many organisms. The highest homologies were 50% to the gor gene of Pseudomonas aeruginosa (43), 49% to TftF of Burkholderia cepacia, involved in the degradation of 2,4,5-trichlorophenoxyacetate (12), and 41% to the gor gene of Streptococcus thermophilus (42). The highly conserved amino acids involved in the binding of NADPH, the substrate glutathione disulphide, and the redox-active cysteins (43), were found in the predicted amino acid sequence of ORF4.…”

Section: Resultsmentioning

confidence: 99%

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Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

et al. 1998

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A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4 ), gtdA, fromSphingomonas sp. strain RW5 was cloned and sequenced. ThegtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488–6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km ) and specificity constants (K cat/K m) of the enzyme were determined to be 15 μM and 511 s−1M−1 × 104 for gentisate and 754 μM and 20 s−1 M−1 × 104 for 3,6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

et al. 1998

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“…A 336-bp fragment containing a part of the hydroxyquinol 1,2-dioxygenase gene from Burkholderia cepacia AC1100 was used as a nonspecific DNA probe. The fragment was synthesized by PCR with primers ORF4P1 (5Ј-GCC TGC AGC GGC CCC TTC CAT GTG-3Ј) and ORF4P2 (5Ј-GCC TGC AGC CTC AAG CAT TTG ACC-3Ј) and with pKS100 as a template (13). S1 nuclease analysis.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Activation of the Chlorocatechol Degradative Genes of Ralstonia eutropha NH9

et al. 1999

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Ralstonia eutropha (formerly Alcaligenes eutrophus) NH9 degrades 3-chlorobenzoate via the modifiedortho-cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the cbnABCDoperon encoding the degradative enzymes (including orfX of unknown function) and the divergently transcribed cbnR gene encoding the LysR-type transcriptional regulator of the cbnoperon. The cbnRAB orfXCD gene cluster is nearly identical to the chlorocatechol genes (tcbRCD orfXEF) of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51. Transcriptional fusion studies demonstrated thatcbnR regulates the expression of cbnABCDpositively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively. In vitro transcription assays confirmed that 2-chloro-cis,cis-muconate (2-CM) andcis,cis-muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon. This inducer-recognizing specificity is different from those of the homologous catechol (catBCA) and chlorocatechol (clcABD) operons of Pseudomonas putida, in which only the intermediates of the regulated pathway, CCM forcatBCA and 2-CM for clcABD, act as significant inducers. Specific binding of CbnR protein to the cbnApromoter region was demonstrated by gel shift and DNase I footprinting analysis. In the absence of inducer, a region of ca. 60 bp from position −20 to position −80 upstream of the cbnAtranscriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position −50. Circular permutation gel shift assays demonstrated that CbnR bent the cbnA promoter region to an angle of 78° and that this angle was relaxed to 54° upon the addition of inducer. While a similar relaxation of bending angles upon the addition of inducer molecules observed with the catBCA andclcABD promoters may indicate a conserved transcriptional activation mechanism of ortho-cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.

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“…Surprisingly, the physiological functions of many other bacterial GSTs remain to be established (34). For instance, some GSTs appear to be associated with the metabolism of aromatic compounds, but they are not essential for growth (12).…”

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confidence: 99%

Purification of a Glutathione S -Transferase and a Glutathione Conjugate-Specific Dehydrogenase Involved in Isoprene Metabolism in Rhodococcus sp. Strain AD45

et al. 1999

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A glutathione S-transferase (GST) with activity toward 1,2-epoxy-2-methyl-3-butene (isoprene monoxide) andcis-1,2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1,2-dichloroepoxyethane, withV max values of 66 and 2.4 μmol min−1 mg of protein−1 andKm values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM.1H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD+-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD+ and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.

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Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100

Cited by 73 publications

References 62 publications

Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

Transcriptional Activation of the Chlorocatechol Degradative Genes of Ralstonia eutropha NH9

Purification of a Glutathione S -Transferase and a Glutathione Conjugate-Specific Dehydrogenase Involved in Isoprene Metabolism in Rhodococcus sp. Strain AD45

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