Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4)

Perkins, Edward J.; Lurquin, Paul F.

doi:10.1128/jb.170.12.5669-5672.1988

Cited by 29 publications

(23 citation statements)

References 20 publications

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“…Although TFDA is generally referred to as 2,4-D monooxygenase (9,11,15,23,25,31), we demonstrate that the enzyme is a ferrous iondependent and at-ketoglutarate-dependent dioxygenase that converts a-ketoglutarate to succinate and carbon dioxide concomitant with the conversion of 2,4-D to 2,4-DCP and glyoxylate. * Organic acid determinations.…”

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confidence: 77%

Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase

1993

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The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined. Although the tfd4-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity. Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses a-ketoglutarate as a cosubstrate. The a-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol. By using [1-_4Cla-ketoglutarate, we established that carbon dioxide is the second product derived from ac-ketoglutarate. Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D.The 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation pathway is a paradigm for microbial metabolism of chloroaromatic compounds (10, 27). The scheme for mineralization of 2,4-D was first established in an Arthrobacter species (2,3,18,32,33) and was shown to involve side chain removal, hydroxylation of the resulting 2,4-dichlorophenol (2,4-DCP), opening of the 3,5-dichlorocatechol ring, and subsequent conversion of 2,4-dichloro-cis,cis-muconate to succinate, which is metabolized by the cells. This pathway also is operative in Alcaligenes eutrophus JMP134, in which several of the 2,4-D degradation genes (tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF) have been localized to the transmissible plasmid pJP4 (7-9). Three of the downstream genes, tfd-CDE, exhibit extensive nucleotide sequence similarity to the well-studied chlorocatechol degradation genes clcABD (9a) and tcbCDE (34), and the corresponding chlorocatechol dioxygenase, cycloisomerase, and hydrolase steps are biochemically well understood (9,26). Similarly, the A. eutrophus tfdB gene (24) exhibits significant sequence similarity to the phenol monooxygenase gene pheA (22), and the nicotinamide-dependent, flavin-containing 2,4-DCP hydroxylase that it encodes has been purified and well characterized (17).In contrast to the other tfd genes, the A. eutrophus tfdA gene sequence (31) exhibits no obvious evolutionary relationship to other known sequences and no biochemical characterization of the tfdA gene product (TFDA) has been reported.Here, we identify the complete enzymatic reaction catalyzed by the product of the tfdA gene. Although TFDA is generally referred to as 2,4-D monooxygenase (9,11, 15,23,25,31), we demonstrate that the enzyme is a ferrous iondependent and at-ketoglutarate-dependent dioxygenase that converts a-ketoglutarate to succinate and carbon dioxide concomitant with the conversion of 2,4-D to 2,4-DCP and glyoxylate. Preparation of cell extracts and partial purification of TFDA. Cultures (2 liters) of E. coli JM109(pUS311) were grown overnight at 30 or 370C with moderate aeration. Cells were harvested by centrifugation (6,000 x g) for 20 min and then suspended in 25 ml of 20 mM Tris-1 mM EDTA-0.4 pg of le...

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confidence: 77%

Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase

1993

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“…To complete the sequence of the tfdB and tfdCDEF operons, a 6.2-kb HindIII-SstI fragment of pEP102 was sequenced and joined to that of the 1.5-kb HindIII fragment by sequencing of a 312-base-pair (bp) HindIII fragment from pRD25A. (11,19,30,38). The open arrow depicts the position and direction of transcription of the tfdCDEF operon; the solid arrow indicates the location and orientation of the tfdB operon.…”

Section: Resultsmentioning

confidence: 99%

“…The phagemids pBluescript KS+ (Stratagene) and pTZ18R (United States Biochemical Corp.) and bacteriophage M13mpl8 (25) were used for cloning and construction of sequencing template libraries. Plasmid pRD25A contains an 8.5-kilobase (kb) SstI on HindIII fragment C of pJP4 cloned into pUC19 (30). DNA manipulations were performed as described previously (30 (ORFs) were prepared to analyze the sizes of proteins predicted by the DNA sequence.…”

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confidence: 99%

“…Plasmid pRD25A contains an 8.5-kilobase (kb) SstI on HindIII fragment C of pJP4 cloned into pUC19 (30). DNA manipulations were performed as described previously (30 (ORFs) were prepared to analyze the sizes of proteins predicted by the DNA sequence. All fragments were cloned into the expression vector pBluescript KS + in the correct orientation to express the desired gene from the T7 promoter.…”

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confidence: 99%

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Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4

et al. 1990

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Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorophenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro-and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and t.fdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and ckD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdiF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.Bacteria play a crucial role in the dissimilation of environmental pollutants. In general, the catabolism of aromatic compounds is channeled through catechol intermediates (33). Hence, the ability of an organism to form and catabolize catechols or their derivatives can play a major part in the utilization of an aromatic compound. Comparison of the degradative pathways of different aromatics may help in understanding the evolution of growth substrate specificity.Chlorinated aromatics, such as 2,4-dichlorophenoxyacetic acid (2,4D) and 3-chlorobenzoate (3CBA), are catabolized via a modified ortho pathway (11,12,20,28,34,35; for a review, see reference 33). Pseudomonas putida plasmid pAC27, a deletion derivative of pAC25, confers catabolism of 3CBA (5), whereas Alcaligenes eutrophus JMP134 plasmid pJP4 enables degradation of both 2,4D and 3CBA (10, 11). In both cases, 3CBA appears to be degraded to 3-chlorocatechol by enzymes encoded by chromosomal genes. 2,4D is catabolized by A. eutrophus JMP134 to 2,4-dichlorophenol (2,4DCP) by 2,4D monooxygenase. 2,4DCP is then hydroxylated by 2,4DCP hydroxylase to form 3,5-dichlorocatechol (11; Fig. 1).3-Chlorocatechol is oxidized to maleylacetic acid by enzymes encoded by the clcABD operon in pAC27 (13) and by the tfdCDEF operon of pJP4 (11). The maleylacetic acid is then catabolized by chromosomally encoded enzymes (21). Enzymes involved in this transformation are chlorocatechol 1,2-dioxygenase (catechol 1,2-dioxygenase II or pyrocatechase II; EC 1.13.11.1), encoded b...

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“…Duplication of tfdA has been observed (28), and plasmid pJP4 has been shown to undergo rearrangements when strains containing it are grown on 3-chlorobenzoate (12).…”

Section: Discussionmentioning

confidence: 99%

Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4

1990

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The closely linked structural genes (fdCDEF borne on the 2,4-dichlorophenoxyacetic acid (TFD) catabolic plasmid, pRO101, were cloned into vector pRO2321 as a 12.6-kilobase-pair BamHI C fragment and designated pRO2334. The first gene in this cluster, fdC, encodes chlorocatechol 1,2-dioxygenase and was expressed constitutively. Chlorocatechol 1,2-dioxygenase expression by pRO2334 was repressed in trans by the negative regulatory element, tfdR, on plasmid pRO1949. Derepression of tfdC was achieved when Pseudomonas aeruginosa PA04032 containing both plasmids pRO2334 and pRO1949 was grown in minimal glucose medium containing TFD, 2,4-dichlorophenol, or 4-chlorocatechol, suggesting that TFD and other pathway intermediates can act as inducing compounds. Genetic organization of the tfdCDEF cluster was established by deletion of the tfdC gene, which resulted in the loss of tfdD and tfdE activity, suggesting that genes tfdCDEF are organized in an operon transcribed from the negatively regulated promoter of tfdC. Deletion subcloning of pRO1949 was used to localize tfdR to a 1.2-kilobase-pair BamHI-XhoI region of the BamHI E fragment of plasmid pRO101. The tfdR gene product was shown not to regulate the expression of tfdB, which encodes 2,4-dichlorophenol hydroxylase.

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Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4)

Cited by 29 publications

References 20 publications

Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase

Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase

Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4

Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4

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