The metabolic pathway of 4-aminophenol in Burkholderia sp. strain AK-5 differs from that of aniline and aniline with C-4 substituents

Burkholderia sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with K(m) and V(max) values of 9.6 micro M and 6.8 micro mol min(-1) mg of protein(-1), respectively.     

The Metabolic Pathway of 4-Aminophenol in Burkholderia sp. Strain AK-5 Differs from That of Aniline and Aniline with C-4 Substituents

Burkholderia sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with K_m and V_max values of 9.6 μM and 6.8 μmol min⁻¹ mg of protein⁻¹, respectively.     

Detoxification mechanisms for 1,2,4-benzenetriol employed by a Rhodococcus sp. BPG-8

A Rhodococcus sp. BPG-8 produces 1,2,4-benzenetriol during the transformation of resorcinol by phloroglucinol induced cell-free extract. The oxidation of 1,2,4-benzenetriol to 2-hydroxy-1,4-benzoquinone produces superoxide radicals that may have potential deleterious effects on cellular integrity. It has been shown that both superoxide dismutase (SOD) and catalase retard the autoxidation of 1,2,4-benzenetriol to 2-hydroxy-1,4-benzoquinone. Termination of the free radical chain reaction between superoxide radical and 1,2,4-benzenetriol seems to prevent this autoxidation. A NAD(P)H-dependent reductase appears to convert the 2-hydroxy-1,4-benzoquinone back to 1,2,4-benzenetriol. Both of these mechanisms appear to stabilize 1,2,4-benzenetriol so that it may be cleaved by meta cleavage enzymes. The enzymes responsible for the stabilization of 1,2,4-benzenetriol appear not to be inducible.     

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.     

Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrysosporium.

Under ligninolytic conditions, the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell extracts. The multistep pathway involves the initial reduction of I to yield 2-amino-4-nitrotoluene (II). II is oxidized by MnP to yield 4-nitro-1,2-benzoquinone (XII) and methanol. XII is then reduced to 4-nitro-1,2-hydroquinone (V), and the latter is methylated to 1,2-dimethoxy-4-nitrobenzene (X). 4-Nitro-1,2-hydroquinone (V) is also oxidized by MnP to yield nitrite and 2-hydroxybenzoquinone, which is reduced to form 1,2,4-trihydroxybenzene (VII). 1,2-Dimethoxy-4-nitrobenzene (X) is oxidized by LiP to yield nitrite, methanol, and 2-methoxy-1,4-benzoquinone (VI), which is reduced to form 2-methoxy-1,4-hydroquinone (IX). The latter is oxidized by LiP and MnP to 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (VII). The key intermediate 1,2,4-trihydroxybenzene is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial reduction of a nitroaromatic group generates the peroxidase substrate II. Oxidation of II releases methanol and generates 4-nitro-1,2-benzoquinone (XII), which is recycled by reduction and methylation reactions to regenerate intermediates which are in turn substrates for peroxidase-catalyzed oxidation leading to removal of the second nitro group. Thus, this unique pathway apparently results in the removal of both aromatic nitro groups before ring cleavage takes place.     

Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrysosporium.

Under ligninolytic conditions, the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell extracts. The multistep pathway involves the initial reduction of I to yield 2-amino-4-nitrotoluene (II). II is oxidized by MnP to yield 4-nitro-1,2-benzoquinone (XII) and methanol. XII is then reduced to 4-nitro-1,2-hydroquinone (V), and the latter is methylated to 1,2-dimethoxy-4-nitrobenzene (X). 4-Nitro-1,2-hydroquinone (V) is also oxidized by MnP to yield nitrite and 2-hydroxybenzoquinone, which is reduced to form 1,2,4-trihydroxybenzene (VII). 1,2-Dimethoxy-4-nitrobenzene (X) is oxidized by LiP to yield nitrite, methanol, and 2-methoxy-1,4-benzoquinone (VI), which is reduced to form 2-methoxy-1,4-hydroquinone (IX). The latter is oxidized by LiP and MnP to 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (VII). The key intermediate 1,2,4-trihydroxybenzene is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial reduction of a nitroaromatic group generates the peroxidase substrate II. Oxidation of II releases methanol and generates 4-nitro-1,2-benzoquinone (XII), which is recycled by reduction and methylation reactions to regenerate intermediates which are in turn substrates for peroxidase-catalyzed oxidation leading to removal of the second nitro group. Thus, this unique pathway apparently results in the removal of both aromatic nitro groups before ring cleavage takes place.     

Degradation of 2,4,6-Trichlorophenol by Phanerochaete chrysosporium: Involvement of Reductive Dechlorination

Under secondary metabolic conditions, the lignin-degrading basidiomycete Phanerochaete chrysosporium mineralizes 2,4,6-trichlorophenol. The pathway for the degradation of 2,4,6-trichlorophenol has been elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway is initiated by a LiP- or MnP-catalyzed oxidative dechlorination reaction to produce 2,6-dichloro-1,4-benzoquinone. The quinone is reduced to 2,6-dichloro-1,4-dihydroxybenzene, which is reductively dechlorinated to yield 2-chloro-1,4-dihydroxybenzene. The latter is degraded further by one of two parallel pathways: it either undergoes further reductive dechlorination to yield 1,4-hydroquinone, which is ortho-hydroxylated to produce 1,2,4-trihydroxybenzene, or is hydroxylated to yield 5-chloro-1,2,4-trihydroxybenzene, which is reductively dechlorinated to produce the common key metabolite 1,2,4-trihydroxybenzene. Presumably, the latter is ring cleaved with subsequent degradation to CO₂. In this pathway, the chlorine at C-4 is oxidatively dechlorinated, whereas the other chlorines are removed by a reductive process in which chlorine is replaced by hydrogen. Apparently, all three chlorine atoms are removed prior to ring cleavage. To our knowledge, this is the first reported example of aromatic reductive dechlorination by a eukaryote.     

Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions the white rot basidiomycete Phanerochaete chrysosporium rapidly mineralizes 2,4,5-trichlorophenol. The pathway for degradation of 2,4,5-trichlorophenol was elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway involves cycles of peroxidase-catalyzed oxidative dechlorination reactions followed by quinone reduction reactions to yield the key intermediate 1,2,4,5-tetrahydroxybenzene, which is presumably ring cleaved. In the first step of the pathway, 2,4,5-trichlorophenol is oxidized to 2,5-dichloro-1,4-benzoquinone by either MnP or Lip. 2,5-Dichloro-1,4-benzoquinone is then reduced to 2,5-dichloro-1,4-hydroquinone. The 2,5-dichloro-1,4-hydroquinone is oxidized by MnP to generate 5-chloro-4-hydroxy-1,2-benzoquinone. The orthoquinone is in turn reduced to 5-chloro-1,2,4-trihydroxybenzene. Finally, the 5-chlorotrihydroxybenzene undergoes another cycle of oxidative dechlorination and reduction reactions to generate 1,2,4,5-tetrahydroxybenzene. The latter is presumably ring cleaved, with subsequent degradation to CO2. In this pathway, the substrate is oxidatively dechlorinated by LiP or MnP in a reaction which produces a quinone. The quinone intermediate is recycled by a reduction reaction to regenerate an intermediate which is again a substrate for peroxidase-catalyzed oxidative dechlorination. This pathway apparently results in the removal of all three chlorine atoms before ring cleavage occurs.     

Study of the protein-ubiquinone interaction in succinate-cytochrome C reductase with azido-ubiquinone derivatives

Several azido-ubiquinones have been synthesized for the study of protein-ubiquinone interaction in succinate-cytochrome $\text{c}$ reductase. In the absence of light, azido-ubiquinones are partially effective in restoring enzymatic activity to ubiquinone- and phospholipid-depleted reductase and the binding of azido-ubiquinones can be partially reversed by 5-(10-bromodecyl)-ubiquinone. When 2-azido-3-methoxy-5-geranyl-6-methyl-1,4-benzoquinone reactivated reductase is illuminated with long wavelength UV light, a complete and irreversible inhibition is observed. This specific photo-inactivation, exerted only by 2-azido-3-methoxy-5-geranyl-6-methyl-1,4-benzoquinone, and not by other azido-ubiquinone derivatives, is evidence for the existence of a specific benzoquinone ring binding site in the enzyme.     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

Evidence of Two Oxidative Reaction Steps Initiating Anaerobic Degradation of Resorcinol (1,3-Dihydroxybenzene) by the Denitrifying Bacterium Azoarcus anaerobius

The denitrifying bacterium Azoarcus anaerobius LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy. The anaerobic degradation of this compound was investigated in cell extracts. Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism. Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K₃Fe(CN)₆ as the electron acceptor. HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy. Average specific activities were 60 mU mg of protein⁻¹ for resorcinol hydroxylation and 150 mU mg of protein⁻¹ for HHQ dehydrogenation. Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation. These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.     

Degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9

Abstract The aerobic degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9 was studied. Intact cells of Pseudomonas sp. BN9 grown with 2,4-dihydroxybenzoate oxidized 2,4-dihydroxybenzoate but not salicylate. Cell-free extracts of Pseudomonas sp. BN9 converted 2,4-dihydroxybenzoate after the addition of NAD(P)H. A partially purified protein fraction converted 2,4-dihydroxybenzoate with NADH to 1,2,4-trihydroxybenzene. 1,2,4-Trihydroxybenzene was converted by a 1,2-dioxygenase to maleylpyruvate, which was reduced by a NADH-dependent enzyme to 3-oxoadipate. 2,4-Dihydroxybenzoate 1-monooxygenase, 1,2,4-trihydroxybenzene 1,2-dioxygenase and maleylpyruvate reductase were induced in Pseudomonas sp. BN9 after growth with 2,4-dihydroxybenzoate.     

Protein-ubiquinone interaction in bovine heart mitochondrial succinate-cytochrome c reductase. Synthesis and biological properties of fluorine substituted ubiquinone derivatives.

To investigate the protein-ubiquinone interaction in the bovine heart mitochondrial succinate-cytochrome c reductase region of the respiratory chain, three fluorine substituted ubiquinone derivatives, 2,3-dimethoxy-6-(9'-fluorodecyl)-1,4-benzoquinone (9FQ), 2-methoxy-5-trifluoromethyl-6-decyl-1,4-benzoquinone (TFQ), and 2-methoxy-5-trifluoromethyl-6-(9'-fluorodecyl)-1,4-benzoquinone (9FTFQ), were synthesized. 9FQ was synthesized by radical coupling of Q0 and bis(10-fluoroundecanoyl)peroxide. The latter was prepared by fluorination of undecylenic acid followed by thionylchloride treatment and peroxidation. TFQ was synthesized from 2,2,2-trifluoro-p-cresol by methylation, nitration, reduction, acetylation, nitration, reduction, oxidation, and radical alkylation. 9FTFQ was prepared by the radical alkylation of 2-methoxy-5-trifluoromethyl-1,4-benzoquinone with bis(10-fluoroundecanoyl)peroxide. All three fluoro-Q derivatives are active (greater than 50% the activity of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) when used as electron acceptors for succinate-ubiquinone reductase. However, only 9FQ is active when used as an electron donor for ubiquinol-cytochrome c reductase or as an electron mediator for succinate-cytochrome c reductase. Both TFQ and 9FTFQ are competitive inhibitors for ubiquinol-cytochrome c reductase. A 19FNMR peak-broadening effect was observed for 9FQ when it was reconstituted with ubiquinone-depleted ubiquinol-cytochrome c reductase. A drastic up-field chemical shift was observed for TFQ when it was reconstituted with ubiquinone-depleted reductase. These results indicate that the binding environments of the benzoquinone ring and the alkyl side chain of the Q molecule are different. The strong up-field chemical shift for TFQ, and lack of significant chemical shift for 9FQ, suggest that the benzoquinone ring is bound near the paramagnetic cytochrome b heme.     

Purification and characterization of an intracellular NADH: quinone reductase from Trametes versicolor

Intracellular NADH:quinone reductase involved in degradation of aromatic compounds including lignin was purified and characterized from white rot fungus Trametes versicolor. The activity of quinone reductase was maximal after 3 days of incubation in fungal culture, and the enzyme was purified to homogeneity using ion-exchange, hydrophobic interaction, and gel filtration chromatographies. The purified enzyme has a molecular mass of 41 kDa as determined by SDS-PAGE, and exhibits a broad temperature optimum between 20-40 degrees C , with a pH optimum of 6.0. The enzyme preferred FAD as a cofactor and NADH rather than NADPH as an electron donor. Among quinone compounds tested as substrate, menadione showed the highest enzyme activity followed by 1,4-benzoquinone. The enzyme activity was inhibited by CuSO(4), HgCl(2), MgSO(4), MnSO(4), AgNO(3), dicumarol, KCN, NaN(3), and EDTA. Its Km and Vmax with NADH as an electron donor were 23 microM and 101 mM/mg per min, respectively, and showed a high substrate affinity. Purified quinone reductase could reduce 1,4-benzoquinone to hydroquinone, and induction of this enzyme was higher by 1,4-benzoquinone than those of other quinone compounds.     

Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2′-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to γ-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD⁺, γ-hydroxymuconic semialdehyde was converted to β-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and γ-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to β-ketoadipic acid via γ-hydroxymuconic semialdehyde and maleylacetic acid.     

The plant antifungal isoflavone genistein is metabolized by Armillaria mellea Vahl to give non-fungitoxic products

Abstract

Armillaria mellea, the causal agent of root rot, is a fungal pathogen which proved able to convert the leguminous plant antifungal compound 4′,5,7-trihydroxyisoflavone (genistein) into intermediate metabolites. After suitable periods of incubation, the metabolites were extracted and concentrated from liquid culture media, containing both the isoflavone and the fungus. After purification by column chromatography, the molecular structure of the metabolites was determined by means of mass spectrometry and nuclear magnetic resonance analyses. Five different compounds were identified: 1,3,5-trihydroxybenzene, 4-hydroxyphenylacetic acid, 2,5-dihydroxyphenylacetic acid (homogentisic acid), its lactone 5-hydroxy-2(3H)-benzofuranone, and 1,4-benzoquinone. In vitro experiments showed that while the starting compound, i.e. genistein, has some activity in inhibiting the growth of the fungal pathogen, the degradation products are devoid of any appreciable fungitoxic activity. Moreover, results show that the isoflavone metabolites can be, at least partially, utilized by A. mellea as a carbon source.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K+, Na+, and NH4+, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

Cloning,Sequence Analysis,and Expression in Escherichia coli of the Gene Encoding Monovalent Cation-activated Levodione Reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K⁺, Na⁺, and NH₄ ⁺, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans

The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.