Metabolism of 4-chlorophenol by Azotobacter sp. GP1: Structure of the meta cleavage product of 4-chlorocatechol

Wieser, Marco

doi:10.1016/0378-1097(94)90110-4

Cited by 20 publications

(23 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…strain GP1, to induce the TCP-dehalogenating enzyme suggests that this organism degrades phenol and TCP via different pathways. This agrees with findings obtained by induction experiments with whole cells in the presence and absence of chloramphenicol (14) and with results concerning the metabolism of phenol (32).…”

Section: Discussionsupporting

confidence: 92%

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1

et al. 1997

Self Cite

View full text Add to dashboard Cite

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O 2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl ؊ ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O 2 per mol of TCP and the formation of 1 mol of Cl ؊ ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O 2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H 2 O 2 . The NH 2 -terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.Polychlorinated phenols are widespread environmental pollutants, and their degradation has been the subject of numerous investigations (for reviews, see references 5, 9, 10, and 21). While many different microorganisms with the capacity to degrade these compounds have been isolated, relatively few reports have dealt with the initial step of their degradation. Moreover, the majority of these investigations were performed with whole cells, crude extracts, or partially purified enzymes, and so far only two polychlorophenol-dehalogenating enzymes from Burkholderia (formerly Pseudomonas) cepacia AC1100 (33) and from a Flavobacterium sp. (34) have been purified to homogeneity. Other polychlorophenol dehalogenases were partially purified from an Arthrobacter sp. (23) or found to be membrane-associated enzymes in Rhodococcus chlorophenolicus (26,27) and Mycobacterium fortuitum (28).The dehalogenation of 2,4,6-trichlorophenol (TCP) was detected in crude extracts of Streptomyces rochei 303 (7) and Pseudomonas pickettii (12). The conversion of TCP to 2,6-dichlorohydroquinone was also observed with whole cells from Pseudomonas cepacia AC1100 (25).In this paper, we describe the purification and characterization of a TCP-dehalogenating enzyme from t...

show abstract

Section: Discussionsupporting

confidence: 92%

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1

et al. 1997

Self Cite

View full text Add to dashboard Cite

show abstract

“…This highly reactive acyl chloride, if released from the enzyme, should be hydrolyzed to 2-hydroxyhexa-2,4-dienoic acid, which shows an absorption maximum at 290 nm at pH 7.5 (38). In contrast, the product of the metacleavage of 4-chlorocatechol has been identified as 5-chloro-2-hydroxy-6-oxohexadienoic acid (30,45) and shows the typical behavior of classical meta-cleavage products. The ring fission product of 3-chlorocatechol after a distal extradiol cleavage would be the isomeric compound 3-chloro-2-hydroxy-6-oxohexadienoic acid exhibiting the same chromophor.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6

et al. 1995

View full text Add to dashboard Cite

An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity. DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp. This is the smallest gene encoding an extradiol dioxygenase found until now. Expression of the gene in a T7 expression vector enabled purification of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-and 4-chlorocatechol, and 3-and 4-methylcatechol. Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.A bacterial strain (strain BN6) which is able to degrade amino-and hydroxynaphthalenesulfonates is currently being studied in this laboratory. This strain oxidizes substituted naphthalenesulfonates to the corresponding substituted 1,2-dihydroxynaphthalenes by a reaction catalyzed by a desulfonating dioxygenase. Subsequent metabolism of the substituted 1,2-dihydroxynaphthalenes (1,2-DHNs) to substituted salicylates follows the common naphthalene degradative pathway. Strain BN6 does not mineralize naphthalenesulfonates, since the salicylates are not further oxidized (24,25,33,34). Cloning the genes for the metabolism of these naphthalenesulfonates would facilitate mobilization into salicylate-degrading bacterial strains, thereby accomplishing the complete degradation of naphthalenesulfonates in a single strain. Previously, the 1,2-DHN dioxygenase (DHNDO), which is part of this metabolic pathway, was purified to homogeneity and biochemically characterized. The enzyme oxidized 1,2-DHN, 2,3-dihydroxybiphenyl (2,3-DHBP), and some other aromatic diols (25). In the present study, we attempted to clone the corresponding gene from strain BN6. Although we obtained clones derived from strain BN6 which were able to oxidize 2,3-DHBP, the encoded gene was unexpectedly not involved in the degradation of naphthalenesulfonates. The extradiol dioxygenase (DO) encoded by this clone was examined in the present study. MATERIALS AND METHODSBacterial strains and culture conditions. The isolation and characterization of strain BN6 have been described previously (33). The strain has been deposited at the Deutsche Sammlung von Mikroorganismen, Brunswick, Germany, as DSM 6383. For the isolation of genomic DNA, the strain was grown in nutrient broth. Escherichia coli DH5␣ was the host for construction of the genomic library. E. coli JM 109 was used for subcloning and isolation of DNA for sequencing. Fo...

show abstract

“…© 1998 The Society for Applied Microbiology chlorinated phenols (Steiert and Crawford 1986 ;Li et al 1991 ;Kiyohara et al 1992 ;Wieser et al 1994 ;Fava et al 1995 ;Tomasi et al 1995 ;Bock et al 1996 ;Koh et al 1997). Under anaerobic conditions, CPs, independently from the substitution grade, are often reductively dechlorinated up to phenol in fresh water sediments and by microbial consortia under sulphate-reducing and methanogenic conditions (Zhang and Wiegel 1990 ;Häggblom and Young 1990 ;Turner Togna et al 1995) ; few pure cultures have been shown to degrade CPs anaerobically (Mohn and Kennedy 1992 ;Utkin et al 1995 ;Christiansen and Ahring 1996).…”

Section: Introductionmentioning

confidence: 99%

Degradation of 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration

Andreoni¹,

Baggi²,

Colombo³

et al. 1998

Lett Appl Microbiol

View full text Add to dashboard Cite

A selected mixed culture and a strain of Alcaligenes eutrophus TCP were able to totally degrade 2,4,6‐TCP with stoichiometric release of Cl−. In cultures of Alc. eutrophus TCP, a dioxygenated dichlorinated metabolite was detected after 48 h of incubation. Experiments conducted with soil microcosms gave evidence that : the degradative process had a biotic nature and was accompanied by microbial growth ; the soil used presented an intrinsic degradative capacity versus 2,4,6‐TCP ; the specialized organism used as inoculum was effective in degrading 2,4,6‐TCP in a short time. These results could be utilized for the adoption of appropriate remediation techniques for contaminated soil.

show abstract

Metabolism of 4-chlorophenol by Azotobacter sp. GP1: Structure of the meta cleavage product of 4-chlorocatechol

Cited by 20 publications

References 13 publications

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1

Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6

Degradation of 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration

Contact Info

Product

Resources

About