Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.     

Comparison of two dioxygenases from Pseudomonas putida.

Catechol 2,3-dioxygenase and homoprotocatechuate 2,3-dioxygenase were purified from the same strain of Pseudomonas putida. Molecular weights and subunit sizes were similar, but amino acid compositions showed some marked differences.     

Catabolism of L-tyrosine by the homoprotocatechuate pathway in gram-positive bacteria.

A metabolic pathway for L-tyrosine catabolism involves 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) as substrate for fission of the benzene nucleus. Cell extracts of an organism tentatively identified as a Micrococcus possessed the enzymes required for degrading homoprotocatechuate to succinate and pyruvate, and stoichiometry was established for several of these reactions. When the required coenzymes were added, cell extracts degraded L-tyrosine to the ring-fission product of homoprotocatechuate 2,3-dioxygenase and also converted 4-hydroxyphenylpyruvic acid into 4-hydroxyphenylacetic acid. This compound, in turn, gave stoichiometric amounts of the ring-fission product of homoprotocatechuate by the action of a nicotinamide adenine dinucleotide phosphate-dependent 3-hydroxylase coupled with homoprotocatechuate 2,3-dioxygenase. Evidence is presented that this route for L-tyrosine catabolism is taken by five other gram-positive strains, including Micrococcus lysodeikticus and a species of Bacillus. Five other gram-positive bacteria from other genera employed the alternative homogentisate pathway.     

Purification and characterization of 2'aminobiphenyl-2,3-diol 1,2-dioxygenase from Pseudomonas sp. LD2

Carbazole is a nitrogen-containing heteroaromatic compound that occurs as a widespread and mutagenic environmental pollutant. The 2'aminobiphenyl-2,3-diol 1,2-dioxygenase involved in carbazole degradation was purified to near electrophoretic homogeneity from Pseudomonas sp. LD2 by a combination of ion-exchange chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. This purification was challenging due to the great instability of the enzyme under many standard conditions. The enzyme was also purified to electrophoretic homogeneity from recombinant Escherichia coli expressing the 2'aminobiphenyl-2,3-diol 1,2-dioxygenase-encoding gene cloned from Pseudomonas sp. LD2. The molecular mass of the native enzyme was determined by gel filtration to be 70 kDa. The subunit molecular masses were determined to be 25 and 8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase is an [alpha2beta2] heterotetramer. The optimal temperature and pH for the enzymatic production of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) from 2,3-dihydroxybiphenyl were determined to be 40 degrees C and 8.0, respectively. The maximum observed specific activity on 2,3-dihydroxybiphenyl was 48.1 mmol HOPDA min(-1) mg(-1). This indicated a maximum observed turnover rate of 360,000 molecules HOPDA enz(-1) s(-1). The K'm inhibition constant Ks and Vmax on 2,3 dihydroxybiphenyl were determined to be 5 microM, 37 microM, and 44 mmol min(-1) mg(-1), respectively. These results show that 2'aminobiphenyl-2,3-diol 1,2-dioxygenase is a meta-cleavage enzyme related to the 4,5-protocatechuate dioxygenase family, with comparable purification challenges posed by intrinsic enzyme instability.     

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.     

Expression,purification, and characterization of 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase from carbazole-degrader Pseudomonas resinovorans strain CA10

The two-subunit meta-cleavage enzyme, 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarBaBb), from the carbazole degrader Pseudomonas resinovorans strain CA10 was purified to homogeneity from an Escherichia coli strain carrying the expression vector pUCA503, in which two copies of the carBaBb genes are tandemly linked. SDS-PAGE and gel filtration showed that CarB was a alpha2beta2-heterotetrameric enzyme with subunit molecular masses of approximately 10,000 for CarBa and 29,000 for CarBb. The optimum pH for activity was 8.5 and that of temperature was 35 degrees C. The CarB enzyme had a Km of 14 microM and a kcat/Km of 0.25 microM(-1) s(-1) for 2'-aminobiphenyl-2,3-diol, and the catalytic activities for biphenyl-type catecholic substrates were higher than those for monoaromatic catechol derivatives. The enzyme was originally isolated as a meta-cleavage enzyme for 2'-aminobiphenyl-2,3-diol involved in carbazole degradation, but the enzyme was highly specific for 2,3-dihydroxybiphenyl.     

Involvement of lysosomes in substrate stabilization of tryptophan-2,3-dioxygenase in rat liver.

Administration of tryptophan or hydrocortisone to rats caused a several-fold increase in tryptophan-2,3-dioxygenase activity in the liver. Highly purified lysosomes were isolated from livers of tryptophan- or hydrocortisone-treated animals as well as the control rats. Immunoblotting of lysosomal proteins with anti-tryptophan-2,3-dioxygenase showed 48 kDa band, corresponding to the subunit molecular weight of the enzyme. The relative amount of the immuno-reactive substance in the lysosomes from hydrocortisone-treated rats was 3 times higher than the control while the value in the lysosomes from tryptophan-treated rats was essentially the same as in the control. These results indicate that administration of tryptophan renders cytosolic tryptophan-2,3-dioxygenase less vulnerable to lysosomal uptake and causes an accumulation of the enzyme in the cytosol.     

Theoretical study of the catalytic reaction mechanism of MndD

Manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) is an enzyme taking part in the catabolism of aromatic compounds in the environment. It uses molecular oxygen to perform an extradiol cleavage of the ring of the ortho-dihydroxylated aromatic compound homoprotocatechuate. A theoretical investigation of the reaction path for MndD was performed using hybrid density functional theory with the B3LYP functional, and a catalytic mechanism has been suggested. Models of different size were built from the crystal structure of the enzyme and were used in the search for intermediates and transition states. It was found that the substrate first binds at the active site as a monoanion. Next the dioxygen is bound, forming a hydroperoxo intermediate. The O–O bond, activated in this way undergoes homolytic cleavage leading to an oxyl and then to an extra epoxide radical with subsequent opening of the aromatic ring. The lactone ring is then hydrolyzed by the Mn-bound OH group, and the final product is obtained in the last reaction steps. Alternative reaction paths were considered, and their calculated barriers were found to be higher than for the suggested mechanism. The selectivity between the extra- and intra-cleavage pathways was found to be determined by the barriers for the decay of the radical state.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Purification and properties of pyrocatechase II from Pseudomonas putida strain 87]

Induction of modified ortho-pathway enzymes (catechol 1.2-dioxygenase II, muconate cycloisomerase II, dienelactone hydrolase, and maleylacetate reductase) was found in Pseudomonas putida 87, when 3-chlorobenzoic acid was used as a sole carbon and energy source. Catechol 1.2-dioxygenase II, the key chlorocatechol cleaving enzyme, was purified and characterized. The enzyme molecular mass as determined by gel filtration was 65,000 Da; the minimum molecular mass upon SDS electrophoresis was 33,000 Da. The pH and temperature optima for the enzyme were 7.2-7.8 and 35 degrees C, respectively. The highest stability of catechol 1.2-dioxygenase II upon storage was observed in 50 mM Tris-HCl buffer pH 7.8 at 4 degrees C. The relative values of Vmax for catechol 1.2-dioxygenase II with 3-chloro-, 4-chloro-, and 3.5-dichlorocatechols were 28%, 50%, and 41% of those for catechol. The enzyme affinity for chlorocatechols was 3-9 times higher than for methylcatechols and 10-20 times higher than for unsubstituted catechol.     

Catechol 2,3-dioxygenase from the thermophilic, phenol-degrading Bacillus thermoleovorans strain A2 has unexpected low thermal stability

Catechol 2,3-dioxygenase from the thermophilic Bacillus thermoleovorans A2 was purified and characterized. The catechol 2,3-dioxygenase has a molecular mass of 135 000 Da and consists of four identical subunits of 34 700 Da. One iron per enzyme subunit was detected using atom absorption spectroscopy. Enzyme activity was not inhibited by EDTA, suggesting that the iron is tightly bound. Addition of hydrogen peroxide to the enzyme completely destroyed activity, indicating that the iron was in the divalent state. The isoelectric point of the enzyme was 4.8. The enzyme displayed optimal activity at pH 7.2 and 70°C. The half-life of the catechol 2,3-dioxygenase at the optimum temperature was 1.5 min under aerobic conditions and 10 min in a nitrogen atmosphere. This stability of the enzyme is comparable to the stability of the enzyme from the mesophilic Pseudomonas putida mt-2. The stability of the cloned enzyme in E. coli extracts was identical to the stability in wild-type extracts, suggesting that no stabilizing factors were present in Bacillus thermoleovorans A2 In whole cells the half-life of the enzyme at 70°C was approximately 26 min, when protein synthesis was disrupted by chloramphenicol; however, the activity remained constant when protein synthesis was not inhibited. From these results we concluded that catechol 2,3-dioxygenase from Bacillus thermoleovorans A2 is not particularly thermostable, but that the organism retains the ability to degrade phenol at high temperatures because of continuous production of this enzyme. Received: October 10, 1998 / Accepted: March 18, 1999     

Degradation of methoxylated aromatic acids by Pseudomonas putida

J.E. TURNER AND N. ALLISON. 1995. A newly-isolated strain of Pseudomonas putida (HVA-1) utilized homovanillic acid as sole carbon and energy source. Homovanillate-grown bacteria oxidized homovanillate and homoprotocatechuate but monohydroxylated and other methoxylated phenylacetic acids were oxidized poorly; methoxy-substituted benzoates were not oxidized. Extracts of homovanillate-grown cells contained homoprotocatechuate 2,3-dioxygenase but the primary homovanillate-degrading enzyme could not be detected. No other methoxylated phenylacetic acid supported growth of the organism but vanillate was utilized as a carbon and energy source. When homovanillate-grown cells were used to inoculate media containing vanillate a 26 h lag period occurred before growth commenced. Vanillate-grown bacteria oxidized vanillate and protocatechuate but no significant oxygen uptake was obtained with homovanillate and other phenylacetic acid derivatives. Analysis of pathway intermediates revealed that homovanillate-grown bacteria produced homoprotocatechuate, formaldehyde and the ring-cleavage product 5-carboxymethyl 2-hydroxymuconic semialdehyde (CHMS) when incubated with homovanillate but monohydroxylated or monomethoxylated phenylacetic acids were not detected. These results suggest that homovanillate is degraded directly to the ring-cleavage substrate homoprotocatechuate by an unstable but highly specific demethylase and then undergoes extradiol cleavage to CHMS. It would also appear that the uptake/degradatory pathways for homovanillate and vanillate in this organism are entirely separate and independently controlled. If stabilization of the homovanillate demethylase can be achieved, there is potential for exploiting the substrate specificity of this enzyme in both medical diagnosis and in the paper industry.     

Tetrameric structure and cellular location of catechol 2,3-dioxygenase

Catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the TOL plasmid of Pseudomonas putida (pWWO) was investigated by electron microscopy. Negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. Monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in Escherichia coli (pAW31) and P. putida (pWWO), using the protein A-gold technique carried out as a post-embedding immunoelectron microscopy procedure. Our in situ labeling studies revealed a cytoplasmic location of the catechol 2,3-dioxygenase in both cell types.Abbreviations C23O Catechol 2,3-dioxygenase - 3MB 3 Methylbenzoate - AK1 Anti-C23O-IgG-antibody - G Gold particle     

A novel meta-cleavage dioxygenase that cleaves a carboxyl-group-substituted 2-aminophenol. Purification and characterization of 4-amino-3-hydroxybenzoate 2,3-dioxygenase from Bordetella sp. strain 10d.

A bacterial strain that grew on 4-amino-3-hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4-amino-3-hydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110-fold increase in specific activity. The purified enzyme was characterized as a meta-cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4-amino-3-hydroxybenzoic acid, with the consumption of 1 mol of O2 per mol of substrate. The enzyme was therefore designated as 4-amino-3-hydroxybenzoate 2,3-dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21-kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4-amino-3-hydroxybenzoic acid. The Km and Vmax values for this substrate were 35 micro m and 12 micro mol.min-1.(mg protein)-1, respectively. Of the 2-aminophenols tested, only 4-aminoresorcinol and 6-amino-m-cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2-aminophenol 1,6-dioxygenase, in molecular mass, subunit structure and catalytic properties.     

Degradation of (+/-)-synephrine by Arthrobacter synephrinum. Oxidation of 3,4-dihydroxyphenylacetate to 2-hydroxy-5-carboxymethyl-muconate semialdehyde.

1. Cell-free extracts of Arthrobacter synephrinum catalyse the oxidation of 3,4-dihydroxy-phenylacetate. 2. The product of oxidation was characterized as 2-hydroxy-5-carboxymethylmuconate semialdehyde from its chemical behaviour as well as from nuclear-magnetic-resonance spectra. 3. A 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15) was partially purified from A. synephrinum. 4. The enzyme had a Km of 25 micrometer towards its substrate and exhibited typical Michaelis-Menten kinetics. 5. The enzyme also catalysed the oxidation of 3,4-dihydroxymandelate and 3,4-dihydroxyphenylpropionate, at reaction rates of 0.5 and 0.04 respectively of that for 3,4-dihydroxyphenylacetate. 6. The enzyme was sensitive to treatment with thiol-specific reagents. 7. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography was approx. 282000.     

Immobilization of functionally unstable catechol-2,3-dioxygenase greatly improves operational stability

Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearothermophilus has been immobilized on highly activated glyoxyl agarose beads. The enzyme could be fully immobilized at 4 degrees C and pH 10.05 with a high retention of activity (around 80%). Enzyme immobilized under these conditions showed little increase in thermostability compared with the soluble enzyme, but further incubation of immobilized enzyme at 25 degrees C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibiting a 100-fold increase in stability (c.f. the free enzyme). The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentially independent of protein concentration whereas free enzyme was rapidly inactivated at low protein concentrations. An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catechol 2,3-dioxygenases at protein concentrations of 10 μg/ml. Immobilization increased the 'optimum temperature' for activity by 20 degrees C, retained activity at substrate concentrations where the soluble enzyme was fully inactivated and enhanced the resistance to inactivation during catalysis. These results suggest that the immobilization of the enzyme under controlled conditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and increased the rigidity of the subunit structures.     

Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis.

Gentisate:oxygen 1,2-oxidoreductase (decyclizing) (EC 1.13.11.4; gentisate 1,2-dioxygenase) from Moraxella osloensis was purified to homogeneity as shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 154,000 and gives rise to subunits of molecular weight 40,000 in the presence of sodium dodecyl sulfate. Gentisate 1,2-dioxygenase showed broad substrate specificity and attacked a range of halogen- and alkyl-substituted gentisic acids. Maleylpyruvate, the product formed from gentisate, was degraded by cell extracts supplemented with reduced glutathione, but substituted maleylpyruvates were not attacked under these conditions.     

Isolation, characterization and docking studies of 2,3-dihydroxybiphenyl 1,2-dioxygenase from an activated sludge metagenome

A 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (designated as bphC_meta) was identified in activated sludge metagenome by PCR. This gene shared 99% sequence identity with BphC from Burkholderia xenovorans LB400. The enzyme was purified from recombinant Escherichia coli with a subunit molecular mass of 32 ± 1 kDa. It was optimally active at pH 9.0 and 40°C, using 2,3-dihydroxybiphenyl as a substrate. Activity toward substituted catechols was: 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-chlorocatechol (4-methylcatechol). The prediction made by molecular docking was consistent with the kinetic experimental data, and further explained the substrate preference of BphC_meta. The present study could pave the way for the improved understanding and application of BphCs derived from metagenomes.     

Purification and characterization of catechol 2,3-dioxygenase from the aniline degradation pathway of Acinetobacter sp. YAA and its mutant enzyme, which resists substrate inhibition

Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 microM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 microM, while the mutant enzyme loosened substrate inhibition.     

3,4-Dihydroxyphenylacetate 2,3-dioxygenase. A manganese(II) dioxygenase from Bacillus brevis

3,4-Dihydroxyphenylacetate 2,3-dioxygenase, an enzyme which catalyzes the extradiol cleavage of catechols, has been purified from Bacillus brevis. Like other extradiol-cleaving dioxygenases, this enzyme has a molecular weight of 140,000 with four subunits of 36,000 each. Unlike the other enzymes, this dioxygenase is not activated by added ferrous ion, not inhibited by cyanide or diethyldithiocarbamate, and not inactivated by H2O2. X-ray fluorescence and atomic absorption analyses show the enzyme to contain approximately 2 g atoms of manganese per mol of protein. EPR spectra are consistent with a manganese(II) center in an environment of low symmetry. This is the first report of an oxygen-activating manganese enzyme.