Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols

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.     

Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45. A highly associated SDS-stable enzyme catalyzing an intramolecular transfer of hydroxy groups.

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.     

Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45.

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy. The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino-phenol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography. A single 33-kDa polypeptide was detected by denaturing gel electrophoresis. The size of the native protein was estimated to be 30 kDa by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM. The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM. Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene. Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. The products and intermediates are consistent with two two-electron reductions of the parent compound. Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.     

Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups

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 H(2)(18)O did not indicate any (18)O-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 (18)O 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.     

Oxidative Pathway for the Biodegradation of Nitrobenzene by Comamonas sp. Strain JS765

Previous studies have shown that the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 proceeds by the reduction of nitrobenzene through nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage. We report here the isolation of a Comamonas sp. that uses an oxidative pathway for the complete mineralization of nitrobenzene. The isolate, designated strain JS765, uses nitrobenzene as a sole source of carbon, nitrogen, and energy. Nitrobenzene-grown cells oxidized nitrobenzene, with the stoichiometric release of nitrite. Extracts of nitrobenzene-grown JS765 showed high levels of catechol 2,3-dioxygenase activity that were not abolished by heating the cell extracts to 60(deg)C for 10 min. The ring cleavage product had an absorbance maximum at 375 nm, consistent with that of 2-hydroxymuconic semialdehyde. Both NAD-dependent dehydrogenase and NAD-independent hydrolase activities towards 2-hydroxymuconic semialdehyde were induced in extracts of nitrobenzene-grown cells. Catechol accumulated in the reaction mixture when cells preincubated with 3-chlorocatechol were incubated with nitrobenzene. Conversion of nitrobenzene to catechol by induced cells in the presence of 3-chlorocatechol and (sup18)O(inf2) demonstrated the simultaneous incorporation of two atoms of oxygen, which indicated that the initial reaction was dioxygenation. The results indicate that the catabolic pathway involves an initial dioxygenase attack on nitrobenzene with the release of nitrite and formation of catechol, which is subsequently degraded by a meta cleavage pathway.     

Studies of the catabolic pathway of degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45: removal of the amino group from 2-aminomuconic semialdehyde.

Pseudomonas pseudoalcaligenes JS45 utilizes nitrobenzene as the sole source of nitrogen, carbon, and energy. Previous studies have shown that degradation of nitrobenzene involves the reduction of nitrobenzene to nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. In the present paper, we report the enzymatic reactions responsible for the release of ammonia after ring cleavage. 2-Aminomuconic semialdehyde was oxidized to 2-aminomuconate in the presence of NAD by enzymes in crude extracts. 2-Aminomuconate was subsequently deaminated stoichiometrically to 4-oxalocrotonic acid. No cofactors are required for the deamination. Two enzymes, 2-aminomuconic semialdehyde dehydrogenase and a novel 2-aminomuconate deaminase, distinguished by partial purification of the crude extracts, catalyzed the two reactions. 4-Oxalocrotonic acid was further degraded to pyruvate and acetaldehyde. The key enzyme, 2-aminomuconate deaminase, catalyzed the hydrolytic deamination that released ammonia, which served as the nitrogen source for growth of the organism.     

Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonas putida ZWL73

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.     

Production of 2-amino-5-phenoxyphenol from 4-nitrobiphenyl ether using nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45

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     

Bacterial Conversion of Hydroxylamino Aromatic Compounds by both Lyase and Mutase Enzymes Involves Intramolecular Transfer of Hydroxyl Groups

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.     

Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes.

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.     

Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis

The pheA gene encoding the bifunctional P-protein (chorismate mutase:prephenate dehydratase) was cloned from Pseudomonas stutzeri and sequenced. This is the first gene of phenylalanine biosynthesis to be cloned and sequenced from Pseudomonas. The pheA gene was expressed in Escherichia coli, allowing complementation of an E. coli pheA auxotroph. The enzymic and physical properties of the P-protein from a recombinant E. coli auxotroph expressing the pheA gene were identical to those of the native enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeri pheA gene was 1095 base pairs in length, predicting a 365-residue protein product with an Mr of 40,844. Codon usage in the P. stutzeri pheA gene was similar to that of Pseudomonas aeruginosa but unusual in that cytosine and guanine were used at nearly equal frequencies in the third codon position. The deduced P-protein product showed sequence homology with peptide sequences of the E. coli P-protein, the N-terminal portion of the E. coli T-protein (chorismate mutase:prephenate dehydrogenase), and the monofunctional prephenate dehydratases of Bacillus subtilis and Corynebacterium glutamicum. A narrow range of values (26-35%) for amino acid matches revealed by pairwise alignments of monofunctional and bifunctional proteins possessing activity for prephenate dehydratase suggests that extensive divergence has occurred between even the nearest phylogenetic lineages.     

Identification,cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.     

A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.     

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.     

类产碱假单胞菌耐热碱性脂肪酶基因的克隆

将类产碱假单胞菌(Pseudomonas pseudoalcaligene)总DNA经Sau3AI部分酶解后的35～50kbDNA片段与经BamHI线性化及CIAP处理过的粘粒pIJ285连接,以大肠杆菌LE392为受体,构建类产碱假单胞菌的基因文库。通过三丁酸甘油酯平板和橄榄油平板法检测克隆子,获得一株具有耐热碱性脂肪酶活性的菌株LE392(pHZ1401)。随后将pHZ1401上的外源DNA片段进行亚克隆,从而获得了具有脂肪酶活性的菌株HB101(pHZ1402)和HB101(pHZ1403),它们分别携带有2.9kb和3.0kb的外源片段。两外源片段约有2kb的重叠区。HB101(pHZ1403)所分泌的脂肪酶活性比HB101(pHZ1402)高4倍,是出发菌的5倍。     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD⁺. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.     

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.