Catalytic Properties of the 3-Chlorocatechol-Oxidizing 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Sphingomonas sp. Strain BN6

The 2,3-dihydroxybiphenyl dioxygenase from Sphingomonas sp. strain BN6 (BphC1-BN6) differs from most other extradiol dioxygenases by its ability to oxidize 3-chlorocatechol to 3-chloro-2-hydroxymuconic semialdehyde by a distal cleavage mechanism. The turnover of different substrates and the effects of various inhibitors on BphC1-BN6 were compared with those of another 2,3-dihydroxybiphenyl dioxygenase from the same strain (BphC2-BN6) as well as with those of the archetypical catechol 2,3-dioxygenase (C23O-mt2) encoded by the TOL plasmid. Cell extracts containing C23O-mt2 or BphC2-BN6 converted the relevant substrates with an almost constant rate for at least 10 min, whereas BphC1-BN6 was inactivated significantly within the first minutes during the turnover of all substrates tested. Furthermore, BphC1-BN6 was much more sensitive than the other two enzymes to inactivation by the Fe(II) ion-chelating compound o-phenanthroline. The reason for inactivation of BphC1-BN6 appeared to be the loss of the weakly bound ferrous ion, which is the cofactor in the catalytic center. A mutant enzyme of BphC1-BN6 constructed by site-directed mutagenesis showed a higher stability to inactivation by o-phenanthroline and an increased catalytic efficiency for the conversion of 2,3-dihydroxybiphenyl and 3-methylcatechol but was still inactivated during substrate oxidation.     

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.     

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.     

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.     

Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1.

A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.     

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.     

Multiplicity of 2,3-dihydroxybiphenyl dioxygenase genes in the Gram-positive polychlorinated biphenyl degrading bacterium Rhodococcus rhodochrous K37

Rhodococcus rhodochrous K37, a Gram-positive bacterium grown under alkaline conditions, was isolated for its ability to metabolize PCBs. Analysis revealed that it has eight genes encoding extradiol dioxygenase, which has 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and these genes were designated bphC1 to bphC8. According to the classification of extradiol dioxygenases [Eltis, L. D., and Bolin, J. T., J. Bacteriol., 178, 5930-5937 (1996)], BphC3 and BphC6 belong to the type II enzyme group. The other six BphCs were classified as members of the type I extradiol dioxygenase group. BphC4 and BphC8 were classified into a new subfamily of type I, family 3. Two linear plasmids, 200 kb and 270 kb in size, were found in K37, and the bphC6 and bphC8 genes were located in the 200 kb linear plasmid. Northern hybridization analysis revealed that the bphC1, bphC2, and bphC7 genes were induced in the presence of testosterone, the bphC6 gene was induced by fluorene, and the bphC8 gene was induced by biphenyl. All eight BphC products exhibited much higher substrate activity for 2,3-dihydroxybiphenyl than for catechol, 3-methylcatechol, or 4-methylcatechol.     

Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis

The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.     

Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol

3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.     

Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of Pseudomonas putida GJ31

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native M_r value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.     

Distal Cleavage of 3-Chlorocatechol by an Extradiol Dioxygenase to 3-Chloro-2-Hydroxymuconic Semialdehyde

A 2,3-dihydroxybiphenyl 1,2-dioxygenase from the naphthalenesulfonate-degrading bacterium Sphingomonas sp. strain BN6 oxidized 3-chlorocatechol to a yellow product with a strongly pH-dependent absorption maximum at 378 nm. A titration curve suggested (de)protonation of an ionizable group with a pK_a of 4.4. The product was isolated, purified, and converted, by treatment with diazomethane, to a dimethyl derivative and, by incubation with ammonium chloride, to a picolinic acid derivative. Mass spectra and ¹H and ¹³C nuclear magnetic resonance (NMR) data for these two derivatives prove a 3-chloro-2-hydroxymuconic semialdehyde structure for the metabolite, resulting from distal (1,6) cleavage of 3-chlorocatechol. 3-Methylcatechol and 2,3-dihydroxybiphenyl are oxidized by this enzyme, in contrast, via proximal (2,3) cleavage.     

Properties of the trihydroxytoluene oxygenase from Burkholderia cepacia R34: an extradiol dioxygenase from the 2,4-dinitrotoluene pathway

Burkholderia cepacia R34 mineralizes 2,4-dinitrotoluene via an oxidative pathway. The initial steps in the degradative pathway lead to formation of 2,4,5-trihydroxytoluene, which serves as the substrate for the ring cleavage dioxygenase. The trihydroxylated substrate differs from the usual substituted catechols found in pathways for aromatic compound degradation. To determine whether the characteristics of the trihydroxytoluene oxygenase reflect the unusual ring cleavage substrate of the 2,4-dinitrotoluene pathway, the gene encoding trihydroxytoluene oxygenase (dntD) was cloned and sequenced, and ring cleavage activity determined from recombinant bacteria carrying the cloned gene. The findings were compared to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT and to other previously described ring cleavage dioxygenases. The comparison revealed that only 60% identity was shared by the two trihydroxytoluene oxygenases, but the amino acid residues involved in cofactor binding, catalysis, and protein folding were conserved in the DntD sequence. The enzyme catalyzed meta-fission of trihydroxytoluene as well as the substrate analogues 1,2,4-benzenetriol, catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and 2,3-dihydroxybiphenyl. However, results from enzyme assays indicated a strong preference for trihydroxytoluene, implying that it was the native substrate for the enzyme. The apparent enzyme specificity, its similarity to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT, and the distant genetic relationship to other ring cleavage enzymes suggest that dntD evolved expressly to carry out trihydroxytoluene transformation.     

Purification, characterization, and substrate specificity of two 2,3-dihydroxybiphenyl 1,2-dioxygenase from Rhodococcus sp. R04, showing their distinct stability at various temperature

The genes of two 2,3-dihydroxybiphenyl 1,2-dioxygenases (BphC1 and BphC2) were obtained from the gene library of Rhodococcus sp. R04. The enzymes have been purified to apparent electrophoretic homogeneity from the cell extracts of the recombinant harboring bphC1 and bphC2. Both BphC1 and BphC2 were hexamers, consisting of six subunits of 35 and 33kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzymes had similar optimal pH (pH 9.0), but different temperatures for their maximum activity (30 degrees C for BphC1, 80 degrees C for BphC2). In addition, they exhibited distinct stability at various temperatures. The enzymes could cleave a wide range of catechols, with 2,3-dihydroxybiphenyl being the optimum substrate for BphC1 and BphC2. BphC1 was inhibited by 2,3-dihydroxybiphenyl, catechol and 3-chlorocatechol, whereas BphC2 showed strong substrate inhibition for all the given substrates. BphC2 exhibited a half-life of 15min at 80 degrees C and 50min at 70 degrees C, making it the most thermostable extradiol dioxygenase studied in mesophilic bacteria. After disruption of bphC1 and bphC2 genes, R04DeltaC1 (bphC1 mutant) delayed the time of their completely eliminating biphenyl another 15h compared with its parent strain R04, but R04DeltaC2 (bphC2 mutant) lost the ability to grow on biphenyl, suggesting that BphC1 plays an assistant role in the degrading of biphenyl by strain R04, while BphC2 is essential for the growth of strain R04 on biphenyl.     

Nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes.

2,3-Dihydroxybiphenyl dioxygenase, which catalyzes ring metacleavage of 2,3-dihydroxybiphenyl, is encoded by the bphC gene of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and T. Miyazaki, J. Bacteriol. 166:392-398, 1986). We determined the nucleotide sequence of a DNA fragment of 2,040 base pairs which included the bphC gene. The fragment included one open reading frame of 912 base pairs to accommodate the enzyme. The predicted processed amino acid sequence of the enzyme subunit consisted of 302 residues, and its 12 NH2-terminal residues were in perfect agreement with those determined for the enzyme. Approximately 10 base pairs upstream from the initiation codon for 2,3-dihydroxybiphenyl dioxygenase, there was a base sequence complementary to the 3' end of the 16S rRNA from Pseudomonas aeruginosa. There was no promoterlike sequence in the region upstream of the bphC gene, but another long open reading frame was present. A putative bphD gene encoding a metacleavage compound-hydrolyzing enzyme was suggested in the region downstream of the bphC gene.     

Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp. strain RHA1 and cloning of the gene.

Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities. An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Microbiol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC. EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined. The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene. The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 was suggested.     

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).     

Isolation and partial characterization of an extradiol non-haem iron dioxygenase which preferentially cleaves 3-methylcatechol.

A purification procedure has been developed for an extradiol dioxygenase expressed in Escherichia coli, which was originally derived from a Pseudomonas putida strain able to grow on toluidine. Physical and kinetic properties of the enzyme have been investigated. The enzyme has a subunit Mr of 33,500 +/- 2000 by SDS/polyacrylamide-gel electrophoresis. Gel filtration indicates a molecular mass under non-denaturing conditions of 120,000 +/- 20,000. The N-terminal sequence (35 residues) of the enzyme has been determined and exhibits 50% identity with other extradiol dioxygenases. Fe(II) is a cofactor of the enzyme, as it is for other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 3-methylcatechol cleaved at a higher rate than catechol or 4-methylcatechol. Km values for these substrates with this enzyme are all around 0.3 microM. The enzyme exhibits a bell-shaped pH profile with pKa values of 6.9 +/- 0.1 and 8.7 +/- 0.1. These results are compared with those found for other extradiol dioxygenases.     

Metabolism of naphthalene by the biphenyl-degrading bacterium Pseudomonas paucimobilis Q1

Pseudomonas paucimobilis Q1 originally isolated as biphenyl degrading organism (Furukawa et al. 1983), was shown to grow with naphthalene. After growth with biphenyl or naphthalene the strain synthesized the same enzyme for the ring cleavage of 2,3-dihydroxybiphenyl or 1,2-dihydroxynaphthalene. The enzyme, although characterized as 2,3-dihydroxybiphenyl dioxygenase (Taira et al. 1988), exhibited considerably higher relative activity with 1,2-dihydroxynaphthalene. These results demonstrate that this enzyme can function both in the naphthalene and biphenyl degradative pathway.Abbreviations DHBP dihydroxybiphenyl - DHBPDO 2,3-dihydroxybiphenyl dioxygenase - DHDHNDH 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase - DHN 1,2-dihydroxynaphthalene - DHNDO 1,2-dihydroxynaphthalene dioxygenase - HBP cis-2-hydroxybenzalpyruvate - HOPDA 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate - PCB polychlorinated biphenyl - 2NS naphthalene-2-sulfonic acid     

Inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase fromPseudomonas sp. strain CB406 by 3,4-dihydroxybiphenyl (4-phenylcatechol)

Summary 3,4-dihydroxybiphenyl is not a substrate for the 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) from biphenyldegradingPseudomonas sp. strain CB406. It acts as both a reversible inhibitor and a potent inactivator of the enzyme. The inactivation process requires the presence of O₂ and can be reversed by the removal of the 3,4-dihydroxybiphenyl followed by incubation of the enzyme in the presence of dithioerythritol and Fe²⁺ under anaerobic conditions. Two other extradiol dioxygenases behave similarly, the catechol 2,3-dioxygenase (BphE) from strain CB406 and the BphC fromPseudomonas sp. strain LB400. The BphC fromP. testosteroni B-356 also did not cleave 3,4-dihydroxybiphenyl but it was not inactivated.Abbreviations C23o Catechol 2,3-dioxygenase - 34DHBP 3,4-dihydroxybiphenyl     

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.