An investigation of the iron-sulphur proteins of benzene dioxygenase from <i>Pseudomonas putida</i> by electron-spin-resonance spectroscopy

Geary, P. J.; Saboowalla, F; Patil, Daulat S.; Cammack, Richard

doi:10.1042/bj2170667

Cited by 81 publications

(46 citation statements)

References 23 publications

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“…Apart from the cluster-bound iron, additional iron also was detected in phthalate dioxygenase (7) and naphthalene dioxygenase (21 (21,74). The dependency of enzyme activity on exogenous Fe2+ tentatively might be explained as the replacement of mononuclear iron lost in the course of the purification procedure by the Fe2+ ions added (7,26,39).…”

Section: Discussionmentioning

confidence: 99%

Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS

1992

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The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS were purified to homogeneity. Yellow component B is a monomer (Mr,37,500) with NADH-acceptor reductase activity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Component B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 mol of iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2. 1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate to catechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogs with electron-withdrawing substituents at the ortho position were transformed preferentially.Chlorobenzoates are key intermediates in the degradative pathway of chlorobiphenyls (25). Due to their good water solubility and low toxicity, chlorobenzoates are favorable model compounds for studying the degradation of halogenated aromatic substances. The ortho-substituted halobenzoates are of special interest because they are more refractory than the other isomers to biodegradation. Steric hindrance and the effect of chlorine atoms on the electron density at the ortho position of the benzene ring were suggested to be responsible for the resistance of 2-halobenzoates (other than 2-fluorobenzoate) to hydroxylation by the benzoate dioxygenase system (50, 52).A number of reports describe the utilization of 2-chlorobenzoate by various Pseudomonas strains (20,33,62,77). Sylvestre et al. (62) suggested an initial attack of 2-chlorobenzoate by a 2-chlorobenzoate dioxygenase in Pseudomonas sp. strain B-300. Engesser and Schulte (20) postulated a 2-chlorobenzoate 1,2-dioxygenase system catalyzing the conversion of 2-chlorobenzoate to catechol in Pseudomonas putida CLB 250. However, these authors did not detect any 2-chlorobenzoate dioxygenase activity in cell extracts.Pseudomonas cepacia 2CBS utilizes 2-chlorobenzoate as a sole source of carbon and energy. In the first step of 2-chlorobenzoate degradation, 2-chlorobenzoate is converted to catechol, which is subject predominantly to metaring cleavage (23). The enzyme catalyzing the initial degradation step was shown to be a two-component dioxygenase system, previously termed 2-chlorobenzoate 1,2-dioxygenase (23).The benzoate dioxygenases of P. putida (arvilla) C-1 (74) and P. putida B13 (27,50,51) and the isofunctional TOL * Corresponding author.plasmid-encoded enzyme toluate 1,2-dioxygenase from P. putida (arvilla) mt-2 (27, 38, 70, 72) are unable to oxygenate 2-chlorobenzoate. Here we investigated the dehalogenating 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS to compare these multicomponent enzyme systems. MATERIALS AND METHODSMaterials. All chemicals were of the highest purity commercially available.Growth of P. cepacia 2CBS and preparation of crude extracts. P. cepacia 2CBS (23) was grown in chloride-free mineral salts medium (22)

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Section: Discussionmentioning

confidence: 99%

Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS

1992

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“…It is interesting to note that this region contains two conserved cysteine residues (at positions 53 and 74, respectively), each of which is followed by a conserved histidine in the vicinity. Benzene dioxygenase ferredoxin (12) and toluene dioxygenase ferredoxin (34) The complete amino acid sequence of the tmoC product was determined in this study (Tables 2 and 3). This polypeptide has a molecular weight of -12,000.…”

Section: Discussionmentioning

confidence: 99%

Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase

et al. 1991

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Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The T4MO enzyme system has broad substrate specificity and provides a new opportunity for biodegradation of toxic compounds and bioconversions. Its known activities include conversion of a variety of phenyl compounds into the phenolic derivatives and the complete degradation of trichloroethylene. We have cloned and characterized a gene cluster from KR1 that determines the T4MO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but not on toluene. An insert consisting of two SaI fragments of identical size (10.2 kb) was shown to complement the mutant for growth on toluene. One of the Sac fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport for the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene.Pseudomonas mendocina KR1 utilizes toluene as a sole carbon and energy source. Toluene is oxidized in this bacterial strain to protocatechuate, which is further oxidized through ortho ring cleavage to form substrates of tricarboxylic acid cycle (37). The initial step of this pathway is distinctively different from those of other known toluene pathways. Earlier studies have shown that in Pseudomonas putida Fl, toluene is initially oxidized to form (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) via a dioxygenation reaction (Fig. 1) (14, 19, 45). Subsequently, it was found that in P. putida mt-2, toluene is initially attacked at the methyl group by a monooxygenase enzyme system to form benzyl alcohol ( Fig. 1) (39). More recent studies have demonstrated that the initial monooxygenation reaction can also occur on the aromatic ring of toluene. In the toluene-degrading bacterium G4, this reaction hydroxylates toluene to form o-cresol ( Fig. 1) (33). However, in P. mendocina KR1, toluene is initially hydroxylated by a toluene-4-monooxygenase (T4MO) system to form p-cresol ( Fig. 1) (36). The T4MO enzyme ...

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“…The intracellular concentration of enzyme is calculated on the basis of an intracellular volume of 6 x 10-16 liters per cell (20). The molar ratio estimation is based on a ferredoxinBED monomer (Mr of 11,939) Amounts of pure ferredoxinBED protein loaded in tracks labeled "Ferredoxin" were 0.4 and 1 ,ug, respectively. The method was that described by Tan and Mason (23).…”

Section: Methodsmentioning

confidence: 99%

“…The latter consists of two dissimilar subunits arranged in an a2,B2 configuration (26). Both the ferredoxinBED and 'SPBED components contain 2Fe-2S clusters, which have been examined by Mossbauer and electron paramagnetic resonance (EPR) spectroscopy (9,11) and which from sequence information (15,18,24) are probably of the Rieske type, with histidine as well as cysteine ligands (17). Although we have some understanding of the function of each individual component, little is known about the interactions involved among the three components in the overall reaction.…”

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confidence: 99%

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2

et al. 1994

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The benzene dioxygenase from Pseudomonas putida MI] is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxinBED. The relative levels of the three components were analyzed by using '251-labelled antibodies, and the functional molar ratio of ISPBED, ferredoxinBED, and reductaseBED was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxinBED was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxinBED and 'SPBED of whole cells. These results demonstrate that the ferredoxinBED component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxinBED was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxinBED protein' and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxinBED had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxinBED is not limiting at the high intracellular concentration (0.48 mM) found in cells.

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An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy

Cited by 81 publications

References 23 publications

Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS

Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS

Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2

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