Degradation of Polychlorinated Biphenyl Metabolites by Naphthalene-Catabolizing Enzymes

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1,2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2,3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl to 2,3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl (3,4-DH-2,2′,5,5′-TCB), respectively. Our data also suggested that purified 1,2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3,4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3,4-DH-2,2′,5,5′-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids.

1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2+ for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobilis Q1. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3- and 3,4-dihydroxybiphenyl, catechol, and 3-methyl- and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalene-degrading bacteria.     

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1, 2-dihydro-1,2-dihydroxynaphathalene dehydrogenase catalyze dehydrogenation of the same range of substrates.

Pseudomonas putida strain G7 cis-1,2-dihydro-1, 2-dihydroxynaphthalene dehydrogenase (NahB) and Comamonas testosteroni strain B-356 cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) were found to be catalytically active towards cis-2,3-dihydro-2,3-dihydroxybiphenyl (specificity factors of 501 and 5850 s-1 mM-1 respectively), cis-1,2-dihydro-1, 2-dihydroxynaphthalene (specificity factors of 204 and 193 s-1 mM-1 respectively) and 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl (specificity factors of 1.6 and 4.9 s-1 mM-1 respectively). A key finding in this work is the capacity of strain B-356 BphB as well as Burkholderia cepacia strain LB400 BphB to catalyze dehydrogenation of 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl which is the metabolite resulting from the catalytic meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl by LB400 biphenyl dioxygenase.     

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

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.     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Crystallization and preliminary crystallographic analysis of a 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas sp. strain KKS102 having polychlorinated biphenyl (PCB)-degrading activity

Crystals have been obtained for a 2,3-dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain KKS1O2. The crystals were grown using both ammonium sulfate and MPD as the precipitating agents. The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 Å. © 1995 Wiley-Liss, Inc.     

Functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of Pseudomonas origin

Abstract The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.     

Bacterial aromatic ring-cleavage enzymes are classified into two different gene families

Dioxygenases that catalyze the cleavage of the aromatic ring are classified into two groups according to their mode of ring fission. Substrates of ring-cleavage dioxygenases usually contain hydroxyl groups on adjacent aromatic carbons, and intradiol enzymes cleave the ring between these two hydroxyl groups. Extradiol enzymes in contrast cleave the ring between one hydroxylated carbon and its adjacent nonhydroxylated carbon. In this study, we determined the complete nucleotide sequence of nahC, the structural gene for 1,2-dihydroxynaphthalene dioxygenase encoded in the NAH7 plasmid of Pseudomonas putida. This enzyme is an extradiol ring-cleavage enzyme that cleaves the first ring of 1,2-dihydroxynaphthalene. The amino acid sequence of the dioxygenase deduced from the DNA sequence demonstrated that the molecular weight of the enzyme is 33,882. This result was in agreement with those of maxicell analyses that showed that the nahC product was a 36-kDa protein. Interestingly, the amino acid sequence of 1,2-dihydroxynaphthalene dioxygenase was 50% homologous with that of 2,3-dihydroxybiphenyl dioxygenase, which catalyzes extradiol cleavage of the first ring of 2,3-dihydroxybiphenyl (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429). The amino acid sequence similarity of 1,2-dihydroxynaphthalene dioxygenase with catechol 2,3-dioxygenase, which is an authentic extradiol dioxygenase, was rather low (16%). However, a statistical analysis by the method of S. B. Needleman and C. D. Wunsch [1970) J. Mol. Biol. 48, 443-453) clearly showed that these two dioxygenases are evolutionarily related. Therefore, these extradiol enzymes are considered as products of the same gene superfamily. From the significant sequence similarity between intradiol enzymes, it has been shown (Neidle, E. L., Harnett, C., Bonitz, S., and Ornston, L. N. (1988) J. Bacteriol. 170, 4874-4880) that intradiol enzymes evolved from a common ancestor. Comparison of the amino acid sequence of extradiol enzymes with those of intradiol dioxygenases did not show any significant global or localized similarity.     

Dehalogenation, denitration, dehydroxylation, and angular attack on substituted biphenyls and related compounds by a biphenyl dioxygenase

The attack by the bph-encoded biphenyl dioxygenase of Burkholderia sp. strain LB400 on a number of symmetrical ortho-substituted biphenyls or quasi ortho-substituted biphenyl analogues has been investigated. 2,2'-Difluoro-, 2,2'-dibromo-, 2,2'-dinitro-, and 2,2'-dihydroxybiphenyl were accepted as substrates. Dioxygenation of all of these compounds showed a strong preference for the semisubstituted pair of vicinal ortho and meta carbons, leading to the formation of 2'-substituted 2,3-dihydroxybiphenyls by subsequent elimination of HX (X = F, Br, NO(2), or OH). All of these products were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenases of Burkholderia sp. strain LB400 or of Rhodococcus globerulus P6. Dibenzofuran and dibenzodioxin, which may be regarded as analogues of doubly ortho-substituted biphenyls or diphenylethers, respectively, were attacked at the "quasi ortho" carbon (the angular position 4a) and its neighbor. This shows that an aromatic ring-hydroxylating dioxygenase of class IIB is able to attack angular carbons. The catechols formed, 2,3,2'-trihydroxybiphenyl and 2,3,2'-trihydroxydiphenylether, were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenase. While angular attack by the biphenyl dioxygenase was the main route of dibenzodioxin oxidation, lateral dioxygenation leading to dihydrodiols was the major reaction with dibenzofuran. These results indicate that this enzyme is capable of hydroxylating ortho or angular carbons carrying a variety of substituents which exert electron-withdrawing inductive effects. They also support the view that the conversions of phenols into catechols by ring-hydroxylating dioxygenases, such as the transformation of 2,2'-dihydroxybiphenyl into 2,3,2'-trihydroxybiphenyl, are the results of di- rather than of monooxygenations. Lateral dioxygenation of dibenzofuran and subsequent dehydrogenation and extradiol dioxygenation by a number of biphenyl-degrading strains yielded intensely colored dead-end products. Thus, dibenzofuran can be a useful chromogenic indicator for the activity of the first three enzymes of biphenyl catabolic pathways.     

Beijerinckia sp strain B1: a strain by any other name . . . . .

Beijerinckia sp strain B1 grows with biphenyl as its sole source of carbon and energy. A mutant, strain B8/36, oxidized biphenyl to cis-(2S,3R)-dihydroxy-l-phenylcyclohexa-4,6-diene (cis-biphenyl dihydrodiol). Strain B8/36 oxidized anthracene, phenanthrene, benz[a]anthracene and benzo[a]pyrene to cis-dihydrodiols. Other substrates oxidized to cis-dihydrodiols were dibenzofuran, dibenzothiophene and dibenzo-p-dioxin. Biphenyl dioxygenase activity was observed in cells of Beijerinckia B1 and B8/36 after growth in the presence of biphenyl, m-, p-xylene and salicylate. Recent studies have led to the reclassification of Beijerinckia B1 as Sphingomonas yanoikuyae strain B1. Subsequent biotransformation studies showed that S. yanoikuyae B8/36 oxidized chrysene to a bis-cis-diol with hydroxyl substituents at the 3,4- and 9,10-positions. Dihydronaphthalene was oxidized to cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, naphthalene, cis-1,2-dihydroxy-1,2-dihydronaphthalene and 2-hydroxy-1,2-dihydronaphthalene. Anisole and phenetole were oxidized to phenol. Thus the S. yanoikuyae biphenyl dioxygenase catalyzes cis-dihydroxylation, benzylic monohydroxylation, desaturation and dealkylation reactions. To date, the genes encoding biphenyl dioxygenase have not been cloned. However, the nucleotide sequence of a S. yanoikuyaeB1 DNA fragment contains five different α subunits as determined by conserved amino acids coordinating iron in a Rieske [2Fe-2S] center and mononuclear iron at the catalytic site. The specific role of the different putative oxygenases in biotransformation reactions catalyzed by S. yanoikuyae is not known and presents an exciting challenge for future studies. Received 29 May 1999/ Accepted in revised form 23 June 1999     

Characterization of a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a polychlorinated biphenyl- and naphthalene-degrading Bacillus sp. JF8

A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 +/- 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 degrees C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 degrees C and 81 min at 75 degrees C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0-4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mm 2,3-dihydroxybiphenyl. A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 microm for 2,3-dihydroxybiphenyl (at 60 degrees C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme.     

Conversion of substituted naphthalenesulfonates by Pseudomonas sp. BN6

The range of substituted naphthalenesulfonates which are metabolized by Pseudomonas sp. BN6 were investigated. Resting cells from strain BN6 oxidized 1- and 2-naphthalenesulfonate, 1-hydroxynaphthalene-2-sulfonate, 2,6-naphthalenedisulfonate and all monosulfonated naphthalene-2-sulfonates which carry one or two substitutents in the positions 4-, 5-, 6-, 7- or 8- of the naphthalene ring-system. With the exception of (substituted) 4- or 5-amino- and 4-hydroxynaphthalene-2-sulfonates these compounds were converted to the corresponding salicylates. Strain BN6 did not oxidize substituted naphthalene-1-sulfonates, 3-substituted naphthalenesulfonates and substituted naphthalenedisulfonates. Turnover of 4-amino- or 4-hydroxynaphthalene-2-sulfonates resulted in the accumulation of the corresponding naphthoquinones in the culture medium. Thus, degradation of 4-amino- and 4-hydroxynaphthalenesulfonates was restricted by the rapid autoxidation of the substituted 1,2-dihydroxynaphthalenes formed as metabolites. Catabolic activities of strain BN6 for naphthalenesulfonates were induced by salicylate, 3- or 6-hydroxysalicylate, and 3-, 4- or 5-aminosalicylate but not by 4- and 5-hydroxysalicylate. All naphthalenesulfonates that were not converted into the corresponding salicylates, were found to be inefficient as effectors. It was therefore concluded that (substituted) salicylates are the inducers of the relevant enzymes. The degradation of 2-naphthalene-sulfonate by a pure culture of strain BN6 was prevented by the toxicity of the dead-end product salicylate. Substituted salicylates were less toxic and allowed growth of strain BN6 in axenic culture with various substituted naphthalenesulfonates.Abbreviations AB aminobenzoate - ANS aminonaphthalenesulfonate - DHN dihydroxynaphthalene - DHNC dihydroxynaphthalene-carboxylate - DHNDO 1,2-dihydroxynaphthalene dioxygenase - HBPA 2-hydroxybenzalpyruvate aldolase - HNS hydroxynaphthalenesulfonate - HS hydroxysalicylate - Ind-C indolecarboxylate - Ind-S indolesulfonate - MANS N-methylaminonaphthalenesulfonate - NC naphthalenecarboxylate - NDS naphthalenedisulfonate - NQ naphthoquinone - NS naphthalenesulfonate - NSDO naphthalenesulfonate dioxygenase - R_t retention time - SADH salicylaldehyde dehydrogenase - THN trihydroxynaphthalene (hydroxy-1,2-dihydroxynaphthalene)     

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.     

Purification and partial characterization of the extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase,in the fluorene degradation pathway from Rhodococcus sp. strain DFA3

Type II extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α₄β₄ heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe²⁺ increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2′-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The K_m and k_cat/K_m values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 μM and 1.5 × 10^?2 μM^?1sec^?1, and for 2,2′,3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10^?2 μM^?1sec^?1, respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.     

A rapid method for naphtalene dioxygenase assay in whole cells of naphtalene cis-dihydrodiol dehydrogenase blocked Pseudomonas fluoresecens: Screening of potential inducers of dioxygenase activity

A rapid and sensitive photometric method was devised to assay naphthalene dioxygenase in whole cells of Pseudomonas fluorescens NCIMB 40531, a strain derived from a naphthalene-metabolizing isolate by means of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The naphthalene-assimilating pathway of NCIMB 40531 is functionally blocked at the level of cis-1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase and therefore cis-1,2-dihydroxy-1,2-dihydronaphthalene (napthalene dihydrodiol) is accumulated when cultures are supplied with naphthalene.This modified metabolism allowed dioxygenase to be assayed by monitoring product formation. Optimal conditions were selected to give linear optical density vs time curves and reaction rates proportional to dry cell weight (DCW): specific activities of 0.125(+-0.008) mol·min^–1·mgDCW^–1 were consistently obtained in cultures grown on succinate in the presence of naphthalene as inducer. By means of the developed assay, 62 compounds (mainly mono- and bicyclic aromatics) were screened as potential inducers of the dioxygenase activity, when added to the growth medium at the concentration of 100 mg·1^–1: besides naphthalene, the highest activities were induced by 3-methylsalicyclic acid (2-hydroxy-3-methylbenzoic acid), O-acetylsalicylic acid and 5-chlorosalicyclic acid with 0.198, 0.167 and 0.076 mol·min^–1mg DCW^–1, respectively. Under the conditions used, no detectable dioxygenase activity was induced by salicylic acid, which is recognized as the natural inducer of the enzyme in Pseudomonas.     

Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of 2,3-dihydroxybiphenyl, the third step in the biphenyl degradation pathway. The nucleotide sequence of the Pseudomonas putida OU83 gene bphC, which encodes 2,3-DBPD, was cloned into a plasmid pQE31. The His-tagged 2,3-DBPD produced by a recombinant Escherichia coli strain, SG13009(pREP4)(pAKC1), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 2,3-DBPD construction, carrying a single 6×His tail on the N-terminal of the polypeptide, was active. SDS-PAGE analysis of the purified active 2,3-DBPD gave a single band of 34 kDa; this is in agreement with the size of the bphC coding region. The K_m for 2,3-dihydroxybiphenyl was 14.5±2 μM. The enzyme activity was enhanced by ferrous ion but inhibited by ferric ion. The enzyme activity was inhibited by thiol-blocking reagents and heavy metals HgCl₂, CuSO₄, NiSO₄, and CdCl₂. The yield was much higher and the time required to purify recombinant 2,3-DBPD from clone pAKC1 was faster than by the conventional chromatography procedures.     

Purification and properties of 2,3-dihydroxybiphenyl dioxygenase from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes and Pseudomonas aeruginosa carrying the cloned bphC gene.

2,3-Dihydroxybiphenyl dioxygenase, involved in biphenyl and polychlorinated biphenyl degradation, was purified from cell extracts of polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 and Pseudomonas aeruginosa PAO1161 carrying the cloned bphC gene (encoding 2,3-dihydroxybiphenyl dioxygenase). The purified enzyme contained ferrous iron as a prosthetic group. The specific activities decreased with the loss of ferrous iron from the enzyme, and the activity was restored by incubation with ferrous iron in the presence of cysteine. Addition of ferric iron caused the complete inactivation of the enzyme. The molecular weight was estimated to be 250,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band with a molecular weight of 31,000, indicating that the enzyme consists of eight identical subunits. The enzyme was specific only for 2,3-dihydroxybiphenyl with a Km value of 87 microM. No significant activity was observed for 3,4-dihydroxybiphenyl, catechol, or 3-methyl- and 4-methylcatechol. The molecular weight, subunit structure, ferrous iron requirement, and NH2-terminal sequence (starting with serine up to 12 residues) were the same between the two enzymes obtained from KF707 and PAO1161 (bphC).     

Substrate specificity and expression of three 2,3-dihydroxybiphenyl 1,2-dioxygenases from Rhodococcus globerulus strain P6

Rhodococcus globerulus strain P6 contains at least three genes, bphC1, bphC2, and bphC3, coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases; the latter two specify enzymes of the family of one-domain extradiol dioxygenases. In order to assess the importance of these different isoenzymes for the broad catabolic activity of this organism towards the degradation of polychlorinated biphenyls (PCBs), the capacities of recombinant enzymes expressed in Escherichia coli to transform different chlorosubstituted dihydroxybiphenyls formed by the action of R. globerulus P6 biphenyl dioxygenase and biphenyl 2,3-dihydrodiol dehydrogenase were determined. Whereas both BphC2 and BphC3 showed similar activities for 2,3-dihydroxybiphenyl and all monochlorinated 2,3-dihydroxybiphenyls, BphC1 exhibited only weak activity for 2'-chloro-2,3-dihydroxybiphenyl. More highly chlorinated 2'-chlorosubstituted 2,3-dihydroxybiphenyls were also transformed at high rates by BphC2 and BphC3 but not BphC1. In R. globerulus P6, BphC2 was constitutively expressed, BphC1 expression was induced during growth on biphenyl, and BphC3 was not expressed at significant levels under the experimental conditions. Although we cannot rule out the expression of BphC3 under certain environmental conditions, it seems that the contrasting substrate specificities of BphC1 and BphC2 contribute significantly to the versatile PCB-degrading phenotype of R. globerulus P6.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.