Characterization of 2,6-Dichloro-p-hydroquinone 1,2-Dioxygenase (PcpA) of Sphingomonas chlorophenolica ATCC 39723

Xun, Luying; Bohuslavek, Jan; Cai, Mian

doi:10.1006/bbrc.1999.1805

Cited by 65 publications

(57 citation statements)

References 18 publications

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“…Supernatants of the PCP-degrading cultures were also analyzed for the accumulation of TeCH, DiCH, and 2-CMA by using previously described HPLC methods (29,40). 2-CMA was prepared by using PcpA to convert DiCH to 2-CMA (33).…”

Section: Methodsmentioning

confidence: 99%

“…Cells that were grown in GMS medium and either induced by PCP (75 M) or not induced were harvested by centrifugation. Cell extracts were obtained by using a French pressure cell as previously described (33) and were stored at Ϫ80°C. Protein concentrations were determined with a dye reagent (Bio-Rad) by using bovine serum albumin as a standard.…”

Section: Methodsmentioning

confidence: 99%

“…Protein concentrations were determined with a dye reagent (Bio-Rad) by using bovine serum albumin as a standard. The activities of PcpB, PcpC, and PcpA were determined by previously described methods (33,35,40). PcpE (2-CMA reductase) activity was determined spectrophotometrically by monitoring NADH oxidation at 340 nm in the presence of 2-CMA in 20 mM potassium phosphate buffer (pH 7.0) at 24°C (27).…”

Section: Methodsmentioning

confidence: 99%

“…TeCH reductive dehalogenase (PcpC) converts TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone (DiCH) by reductive dechlorination (20,39,40). DiCH is subject to ring cleavage by DiCH 1,2-dioxygenase (PcpA), producing 2-chloromaleylacetate (2-CMA) (19,33). The corresponding genes, pcpB, pcpC, and pcpA, have been individually cloned and sequenced (21,22,36).…”

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confidence: 99%

“…pcpR is a hypothetical LysR-type regulator. Northern hybridization and enzymatic activity analysis suggest that PcpB and PcpA are PCP inducible in strain ATCC 39723 (22,33), while PcpC is constitutively produced (20,40). However, the overall organization and regulation of PCP-degrading genes have not been reported, and the metabolic steps beyond ring cleavage by PcpA have not been characterized.…”

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Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723

2002

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The first three enzymes of the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum (formerly Sphingomonas chlorophenolica) ATCC 39723 have been characterized, and the corresponding genes, pcpA, pcpB, and pcpC, have been individually cloned and sequenced. To search for new genes involved in PCP degradation and map the physical locations of the pcp genes, a 24-kb fragment containing pcpA and pcpC was completely sequenced. A putative LysR-type transcriptional regulator gene, pcpM, and a maleylacetate reductase gene, pcpE, were identified upstream of pcpA. pcpE was found to play a role in PCP degradation. pcpB was not found on the 24-kb fragment. The four gene products PcpB, PcpC, PcpA, and PcpE were responsible for the metabolism of PCP to 3-oxoadipate in ATCC 39723, and inactivational mutation of each gene disrupted the degradation pathway. The organization of the pcp genes is unusual because the four PCP-degrading genes, pcpA, pcpB, pcpC, and pcpE, were found to be located at four discrete locations. Two hypothetical LysR-type regulator genes, pcpM and pcpR, have been identified; pcpM was not required, but pcpR was essential for the induction of pcpB, pcpA, and pcpE. The coinducers of PcpR were PCP and other polychlorinated phenols. The expression of pcpC was constitutive. Thus, the organization and regulation of the genes involved in PCP degradation to 3-oxoadipate were documented.Pentachlorophenol (PCP) has been released into the environment as a wood preservative (8, 13). This compound is a major environmental pollutant due to its toxicity and recalcitrance, and it is regulated as one of the priority pollutants by the U.S. Environmental Protection Agency (16, 30). Microorganisms have been used to remove PCP from the environment (16, 17), and several aerobic PCP-degrading bacteria have been isolated from contaminated soils (7). Sphingobium chlorophenolicum (31) (formerly Sphingomonas chlorophenolica) strain ATCC 39723 is one of the bacteria capable of completely mineralizing PCP (24). The biochemistry of PCP degradation by ATCC 39723 has been extensively studied (Fig. 1). PCP 4-monooxygenase (PcpB) oxidizes PCP to 2,3,5,6-tetrachlorop-hydroquinone (TeCH) (22,35,37,38). TeCH reductive dehalogenase (PcpC) converts TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone (DiCH) by reductive dechlorination (20,39,40). DiCH is subject to ring cleavage by DiCH 1,2-dioxygenase (PcpA), producing 2-chloromaleylacetate (2-CMA) (19, 33). The corresponding genes, pcpB, pcpC, and pcpA, have been individually cloned and sequenced (21,22,36). pcpB was found to be physically linked with two other putative pcp genes, pcpD and pcpR (21). pcpR is a hypothetical LysR-type regulator. Northern hybridization and enzymatic activity analysis suggest that PcpB and PcpA are PCP inducible in strain ATCC 39723 (22, 33), while PcpC is constitutively produced (20,40). However, the overall organization and regulation of PCP-degrading genes have not been reported, and the metabolic steps beyond rin...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723

2002

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2,3‐Dihydroxybiphenyl 1,2‐Dioxygenase

Bolin

Eltis

2004

Handbook of Metalloproteins

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2,3‐Dihydroxybiphenyl 1,2‐dioxygenase (DHBD) is an Fe 2+ ‐dependent extradiol dioxygenase. DHBD catalyzes the extradiol cleavage of 2,3‐dihydroxybiphenyl (DHB) to 2‐hydroxy‐6‐oxo‐6‐phenylhexa‐2,4‐dienoate (HOPDA) incorporating both atoms of dioxygen into the product HOPDA. DHBDs are found in a range of Gram‐negative and Gram‐positive bacteria that aerobically assimilate biphenyl. Amino acid sequences are available for over 50 bacterial extradiol dioxygenases that are evolutionarily related to DHBDs (i.e. type I enzymes); all are involved in the degradation of aromatic compounds. The type I bacterial enzymes may have large (∼33 kDa) or small (∼21 kDa) monomers. DHBD catalyzes the third reaction of the upper bph pathway, which catabolizes biphenyl to benzoate and 2‐hydroxypentadienoate. This pathway also transforms some polychlorinated biphenyls (PCBs). DHBDs can be best expressed heterologously in pseudomonads like Burkholderia sp. strain LB400 or Pseudomonas sp. strain KKS102. Mössbauer and EPR spectroscopies demonstrated the presence of high spin Fe 2+ in purified active preparations of Pseudomonas putida mt‐2 (C23O). High‐resolution crystal structures of the active ferrous form of DHBD from Burkholderia sp. strain LB400 and Pseudomonas sp. strain KKS102 were determined. These octameric DHBDs have 422 point group symmetry. The monomer has an α + ß fold that may be subdivided into superimposable barrel‐like N‐ and C‐terminal half‐molecules. The active site Fe and the substrate binding sites are located in the cavity of the C‐terminal half. The ferrous Fe is bound by five ligands in square pyramidal geometry. The axial ligand is a conserved histidine, and the basal ligands are a second conserved histidine, a conserved monodentate glutamic acid, and two water molecules. X‐ray structures of DHBD in complex with DHB and 3‐methyl catechol are known for both the ferric and ferrous forms. Spectroscopic, mechanistic, and X‐ray structural studies were used to elaborate a plausible reaction mechanism for DHBDs.

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Hydroxyquinol 1,2‐Dioxygenase

Briganti

Ferraroni

2004

Handbook of Metalloproteins

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Hydroxyquinol 1,2‐dioxygenase (1,2‐HQD) is an intradiol dioxygenase that uses a nonheme ferric iron to activate the substrate for an electrophilic attack by molecular oxygen to cleave hydroxyquinol (1,2,4‐trihydroxybenzene) or chlorohydroxyquinols between the vicinal hydroxyls. This enzyme is involved in the aerobic metabolism of chloroaromatic compounds containing two or more chlorine substituents. The article focuses on the structure–function relationship of 1,2‐HQD and the comparison, at the molecular level, of the structural factors responsible for the differential substrate selectivity in known ring‐cleaving intradiol dioxygenases.

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Characterization of 2,6-Dichloro-p-hydroquinone 1,2-Dioxygenase (PcpA) of Sphingomonas chlorophenolica ATCC 39723

Cited by 65 publications

References 18 publications

Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723

Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723

2,3‐Dihydroxybiphenyl 1,2‐Dioxygenase

Hydroxyquinol 1,2‐Dioxygenase

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