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1.
Bacterial Conversion of Hydroxylamino Aromatic Compounds by both Lyase and Mutase Enzymes Involves Intramolecular Transfer of Hydroxyl Groups
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Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp. strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. GC-MS analysis of the reaction products formed in H218O did not indicate any 18O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R. eutropha JMP134. During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp. strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was 18O labeled. The other hydroxyl group in the product must have come from the substrate. The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol. The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups. 相似文献
2.
Hydroxylaminobenzene mutase is the enzyme that converts intermediates formed during initial steps in the degradation of nitrobenzene to a novel ring-fission lower pathway in Pseudomonas pseudoalcaligenes JS45. The mutase catalyzes a rearrangement of hydroxylaminobenzene to 2-aminophenol. The mechanism of the reactions and the properties of the enzymes are unknown. In crude extracts, the hydroxylaminobenzene mutase was stable at SDS concentrations as high as 2%. A procedure including Hitrap-SP, Hitrap-Q and Cu(II)-chelating chromatography was used to partially purify the enzyme from an Escherichia coli clone. The partially purified enzyme was eluted in the void volume of a Superose-12 gel-filtration column even in the presence of 0.05% SDS in 25 mM Tris/HCl buffer, which indicated that it was highly associated. When the enzymatic conversion of hydroxylaminobenzene to 2-aminophenol was carried out in 18O-labeled water, the product did not contain 18O, as determined by GC-MS. The results indicate that the reaction proceeded by intramolecular transfer of the hydroxy group from the nitrogen to the C-2 position of the ring. The mechanism is clearly different from the intermolecular transfer of the hydroxy group in the non-enzymatic Bamberger rearrangement of hydroxylaminobenzene to 4-aminophenol and in the enzymatic hydroxymutation of chorismate to isochorismate. 相似文献
3.
A. Schenzle H. Lenke P. Fischer P. A. Williams H. Knackmuss 《Applied microbiology》1997,63(4):1421-1427
Ralstonia eutropha JMP 134 utilizes 3-nitrophenol as the sole source of nitrogen, carbon, and energy. The entire catabolic pathway of 3-nitrophenol is chromosomally encoded. An initial NADPH-dependent reduction of 3-nitrophenol was found in cell extracts of strain JMP 134. By use of a partially purified 3-nitrophenol nitroreductase from 3-nitrophenol-grown cells, 3-hydroxylaminophenol was identified as the initial reduction product. Resting cells of R. eutropha JMP 134 metabolized 3-nitrophenol to N-acetylaminohydroquinone under anaerobic conditions. With cell extracts, 3-hydroxylaminophenol was converted into aminohydroquinone. This enzyme-mediated transformation corresponds to the acid-catalyzed Bamberger rearrangement. Enzymatic conversion of the analogous hydroxylaminobenzene yields a mixture of 2- and 4-aminophenol. 相似文献
4.
Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols 总被引:1,自引:0,他引:1
The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds. 相似文献
5.
Microbial metabolism of nitroarenes via o-aminophenols requires the participation of two key enzymes, a nitroreductase and an hydroxylaminobenzene mutase. The broad
substrate ranges of the enzymes suggested that they could be used as biocatalysts for the production of substituted o-aminophenols. We have used enzymes from Pseudomonas pseudoalcaligenes JS45 for the conversion of 4-nitrobiphenyl ether to the corresponding o-aminophenol. Partially purified nitrobenzene nitroreductase reduced 4-nitrobiphenyl ether to the corresponding 4-hydroxylaminobiphenyl
ether. Partially purified hydroxylaminobenzene mutase stoichiometrically converted the intermediate to 2-amino-5-phenoxyphenol.
The results indicate that the enzyme system can be applied for the production of o-aminophenols useful as intermediates for synthesis of commercially important materials. Journal of Industrial Microbiology & Biotechnology (2000) 24, 301–305.
Received 13 October 1999/ Accepted in revised form 31 January 2000 相似文献
6.
7.
Accumulation of 2-Aminophenoxazin-3-one-7-Carboxylate during Growth of Pseudomonas putida TW3 on 4-Nitro-Substituted Substrates Requires 4-Hydroxylaminobenzoate Lyase (PnbB)
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Michelle A. Hughes Michael J. Baggs Juma'a al-Dulayymi Mark S. Baird Peter A. Williams 《Applied microbiology》2002,68(10):4965-4970
During growth of Pseudomonas putida strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a λmax of 446 nm. The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC). This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water. It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3. Using the cloned pnb structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC. This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site. 相似文献
8.
Hughes MA Baggs MJ Al-Dulayymi J Baird MS Williams PA 《Applied and environmental microbiology》2002,68(10):4965-4970
During growth of Pseudomonas putida strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a lambda(max) of 446 nm. The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC). This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water. It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3. Using the cloned pnb structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC. This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site. 相似文献
9.
A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway. 相似文献
10.
2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45. 总被引:8,自引:0,他引:8
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Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other. Ring fission then occurs at the bond adjacent to one of the hydroxyl groups. In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzene-degrading strain Pseudomonas pseudoalcaligenes JS45. To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography. The molecular mass determined by gel filtration was 140,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an alpha2beta2 subunit structure. Studies with inhibitors indicated that ferrous iron was the sole cofactor. The Km values for 2-aminophenol and oxygen were 4.2 and 710 microM, respectively. The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol. 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3- and 4-methylcatechol were not substrates. The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases. 相似文献
11.
Xiao Y Wu JF Liu H Wang SJ Liu SJ Zhou NY 《Applied microbiology and biotechnology》2006,73(1):166-171
The genes encoding enzymes involved in the initial reactions during degradation of 4-chloronitrobenzene (4CNB) were characterized from the 4CNB utilizer Pseudomonas
putida ZWL73, in which a partial reductive pathway was adopted. A DNA fragment containing genes coding for chloronitrobenzene nitroreductase (CnbA) and hydroxylaminobenzene mutase (CnbB) were PCR-amplified and subsequently sequenced. These two genes were actively expressed in Escherichia
coli, and recombinant E. coli cells catalyzed the conversion of 4CNB to 2-amino-5-chlorophenol, which is the ring-cleavage substrate in the degradation of 4CNB. Phylogenetic analyses on sequences of chloronitrobenzene nitroreductase and hydroxylaminobenzene mutase revealed that these two enzymes are closely related to the functionally identified nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas strains JS45 and HS12. The nitroreductase from strain ZWL73 showed a higher specific activity toward 4CNB than nitrobenzene (approximately at a ratio of 1.6:1 for the recombinant or 2:1 for the wild type), which is in contrast to the case where the nitroreductase from nitrobenzene utilizers Pseudomonas
pseudoalcaligenes JS45 with an apparently lower specific activity against 4CNB than nitrobenzene (0.16:1) [Kadiyala et al. Appl Environ Microbiol 69:6520–6526, 2003]. This suggests that the nitroreductase from 4-chloronitrobenzene utilizer P. putida ZWL73 may have evolved to prefer chloronitrobenzene to nitrobenzene as its substrate.Y.X. and J.-F.W. equally contributed to this work. 相似文献
12.
In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds. 相似文献
13.
Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by Mycobacterium Strain HL 4-NT-1
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In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds. 相似文献
14.
Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3 总被引:3,自引:0,他引:3
Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated with pnbB but appears to have no function in the pathway. The central pnbR gene is transcribed in the opposite direction to the other four genes. These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in P. putida strain TW3. 相似文献
15.
3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 catalyzes a Bamberger rearrangement
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3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone. To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source. 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide. The pI was determined to be at pH 4.5. In a database search, the NH2-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species. Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hydroxylaminobenzoate, can also serve as substrates for the enzyme. The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement. 相似文献
16.
Takenaka S Setyorini E Kim YJ Murakami S Aoki K 《Bioscience, biotechnology, and biochemistry》2005,69(5):1033-1035
Pseudomonas sp. strain AP-3 grows on benzoate, p-hydroxybenzoate, protocatechuate, and 2-aminophenol as sole carbon and energy source. This strain converted benzoate and p-hydroxybenzoate to catechol and protocatechuate respectively, which were metabolized via the ortho-cleavage pathway. The enzymes responsible for these reactions were shown to be inducible. In contrast, strain AP-3 constitutively expresses the enzymes involved in the metabolism of 2-aminophenol. 相似文献
17.
Biotransformation of hydroxylaminobenzene and aminophenol by Pseudomonas putida 2NP8 cells grown in the presence of 3-nitrophenol 总被引:1,自引:0,他引:1
Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone, N-acetyl-4-aminophenol, N-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia, N-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of P. putida 2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in P. putida 2NP8, in which all of the possible intermediates are postulated. 相似文献
18.
Biotransformation of Hydroxylaminobenzene and Aminophenol by Pseudomonas putida 2NP8 Cells Grown in the Presence of 3-Nitrophenol
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Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone, N-acetyl-4-aminophenol, N-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia, N-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of P. putida 2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in P. putida 2NP8, in which all of the possible intermediates are postulated. 相似文献
19.
Pseudomonas sp. strain JS51 grows on m-nitrobenzoate (m-NBA) with stoichiometric release of nitrite. m-NBA-grown cells oxidized m-NBA and protocatechuate but not 3-hydroxybenzoate, 4-hydroxy-3-nitrobenzoate, 4-nitrocatechol, and 1,2,4-benzenetriol. Protocatechuate accumulated transiently when succinate-grown cells were transferred to media containing m-NBA. Respirometric experiments indicated that the conversion of m-NBA to protocatechuate required 1 mol of oxygen per mol of substrate. Conversions conducted in the presence of 18O2 showed the incorporation of both atoms of molecular oxygen into protocatechuate. Extracts of m-NBA-grown cells cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde. These results provide rigorous proof that m-NBA is initially oxidized by a dioxygenase to produce protocatechuate which is further degraded by a 4,5-dioxygenase. 相似文献
20.
A Pseudomonas pseudoalcaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence of NAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized. These results indicate that the catabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene; each of these steps requires 1 mol of NADPH. An enzyme-mediated Bamberger-like rearrangement converts hydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. The mechanism for release of ammonia and subsequent metabolism are under investigation. 相似文献