PcpA, which is involved in the degradation of pentachlorophenol in <i>Sphingomonas chlorophenolica</i> ATCC39723, is a novel type of ring‐cleavage dioxygenase

Ohtsubo, Yoshiyuki; Miyauchi, Keisuke; Kanda, Kenji; Hatta, Takashi; Kiyohara, Houzo; Senda, Toshiya; Nagata, Yasunobu; Mitsui, Yukio; Takagi, Masamichi

doi:10.1016/s0014-5793(99)01305-8

Cited by 67 publications

(52 citation statements)

References 22 publications

(22 reference statements)

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“…Rather, 5-CHQ is further dechlorinated to HQ before HQ 1,2-dioxygenase (TftH) oxidizes it to maleylacetate (12,51). Sphingomonas chlorophenolica ATCC 39723 degrades pentachlorophenol and 2,4,6-TCP to 2,6-DiCH that is directly oxidized to 2-chloromaleylacetate by 2,6-DiCH 1,2-dioxygenase (36,48). Therefore, S. chlorophenolica ATCC 39723 and R. eutropha JMP134 degrade 2,4,6-TCP through two different pathways.…”

Section: Discussionmentioning

confidence: 99%

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

2002

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Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH 2 )-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH 2 -utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH 2 -utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH 2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.

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Section: Discussionmentioning

confidence: 99%

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

2002

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“…Because the putative orf3 gene product showed significant similarity to the novel type of ring cleavage dioxygenases, PcpA and LinE (96 and 53% amino acid identity, respectively), we renamed orf3 linEb. Substrates of PcpA and LinE are HQ-type compounds, i.e., aromatic compounds with two hydroxyl groups at the para position (18,27,40). This type of dioxygenase shows a low degree of similarity to meta cleavage dioxygenases, such as 2,3-dihydroxybiphenyl 1,2-dioxygenases (BphC) (18).…”

Section: Isolation Of a Ut26 Mutant Deficient In ␥-Hch Utilizationmentioning

confidence: 99%

Identification and Characterization of Genes Involved in the Downstream Degradation Pathway of γ-Hexachlorocyclohexane in Sphingomonas paucimobilis UT26

et al. 2005

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Sphingomonas paucimobilis UT26 utilizes ␥-hexachlorocyclohexane (␥-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of ␥-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of ␥-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for ␥-HCH utilization.Sphingomonas paucimobilis UT26 utilizes the halogenated organic insecticide ␥-hexachlorocyclohexane (␥-HCH; also called ␥-BHC and lindane) as a sole source of carbon and energy under aerobic conditions (7). UT26 degrades ␥-HCH through the pathway shown in Fig. 1 (25). ␥-HCH is converted to 2,5-dichlorohydroquinone (2,5-DCHQ) by sequential reactions catalyzed by LinA (␥-HCH dehydrochlorinase) (8,20,26,37), LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene chlorohydrolase) (21, 24), and LinC (2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase) (22). 2,5-DCHQ is then dechlorinated to chlorohydroquinone (CHQ) by LinD (2,5-DCHQ dechlorinase) (17), and CHQ is further transformed to maleylacetate (MA) by LinE (CHQ 1,2-dioxygenase) (18). LinE is the first reported enzyme that prefers hydroquinone (HQ)-type compounds, i.e., aromatic compounds with two hydroxyl groups at the para position, as its substrate to catechol (18). The linA, linB, and linC genes for the upstream pathway are dispersed on the UT26 genome and are constitutively expressed (25). On the other hand, the linD and linE genes form an operon and their expression is induced by a LysR-type transcriptional regulator (LTTR), LinR, in the presence of HQ-type compounds, such as CHQ, HQ, and 2,5-DCHQ (19).By a series of reactions catalyzed by LinA to LinE, ␥-HCH is converted to MA (Fig. 1). MA is known as one of the major intermediates in the degradation of aromatic compounds, and it is usually converted to ␤-ketoadipate by MA reductase. ␤-Ketoadipate is metabolized by the ␤-ketoadipate degradation pathway widely distributed in soil bacteria and fungi (5, 9, 13, 33, 34). In this study, we identified and characterized a gene, designated linF, encoding MA reductase, which was necessary for the assimilation of ␥-HCH in UT26. We further characterized a gene encoding a LinE homologue that was located close to linF. MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli cells were grown at 37°C in ...

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“…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%

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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PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring‐cleavage dioxygenase

Cited by 67 publications

References 22 publications

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

Identification and Characterization of Genes Involved in the Downstream Degradation Pathway of γ-Hexachlorocyclohexane in Sphingomonas paucimobilis UT26

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

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