Zhongqi He

Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from Pseudomonas pseudoalcaligenes JS45

ABSTRACT Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (habA and habB), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85°C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60°C. HAB mutase activity in extracts of P. pseudoalcaligenesJS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and thehabB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli, reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosisH37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. colicontaining M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB, as well as downstream ofhabB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.

Genetic and biochemical comparison of 2-aminophenol 1,6-dioxygenase of Pseudomonas pseudoalcaligenes JS45 to meta-cleavage dioxygenases: divergent evolution of 2-aminophenol meta-cleavage pathway

Abstract Nitrobenzene is degraded to pyruvate and acetaldehyde by Pseudomonas pseudoalcaligenes JS45 via a reductive pathway, and by Comamonas sp. JS765 via an oxidative pathway. Although the initial reactions in the degradation of nitrobenzene by the two bacteria are totally different, the lower pathways are similar and converge at the level of 4-oxalocrotonate. In order to further investigate the biochemical properties and reveal the evolutionary relationships between the two lower pathways, the genes encoding the 2-aminophenol 1,6-dioxygenase were cloned and sequenced. 2-Aminophenol 1,6-dioxygenase from P. pseudoalcaligenes JS45 and catechol 2,3-dioxygenase from Comamonas sp. JS765 were able to act on both catechol and 2-aminophenol, but catechol was a suicide substrate of 2-aminophenol 1,6-dioxygenase. The activity of 2-aminophenol 1,6-dioxygenase was restored after removal of catechol and incubation with ascorbate and FeCl2. Both the α-subunit (AmnA) and the β-subunit (AmnB) of the dioxygenase from P. pseudoalcaligenes JS45 show a high degree of identity to the corresponding subunits of the ring-fission dioxygenase from Pseudomonas sp. AP-3: 67% for the α-subunit, and 84% for the β-subunit. Sequence similarity studies suggest that the β-subunits of both 2-aminophenol 1,6-dioxygenases are distantly related to homoprotocatechuate 2,3-dioxygenase from Escherichia coli strains W and C and then to catechol 2,3-dioxygenase from Alcaligenes eutrophus. Four active-site-relevant histidines are conserved in AmnB, but not in AmnA. The lack of conserved histidines indicates the absence of an Fe2+ binding site in AmnA, which explains the previous observations of only approximately one Fe2+ per two subunits in the 2-aminophenol 1,6-dioxygenases from P. pseudoalcaligenes JS45. The 2-aminophenol 1,6-dioxygenase genes are located upstream of the 2-aminomuconic semialdehyde dehydrogenase gene, and a putative member of the YjgF protein family is upstream of the dioxygenase genes. Transcriptional analysis indicates that the YjgF-like protein, 2-aminophenol 1,6-dioxygenase, and 2-aminomuconic semialdehyde dehydrogenase are coordinately transcribed. A putative ORF similar to part of the RNA helicase genes is downstream of the dehydrogenase gene. Both the novel organization of the genes and the phylogeny of the dioxygenases and dehydrogenase indicate that the 2-aminophenol pathway in P. pseudoalcaligenes JS45 represents an example of a distant divergent evolution of meta-cleavage pathways.

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

ABSTRACT 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.

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.

One-step production of picolinic acids from 2-aminophenols catalyzed by 2-aminophenol 1,6-dioxygenase

Picolinic acids have been synthesized previously from catechols by the action of catechol 2,3-dioxygenase and a subsequent chemical reaction in the presence of ammonia. 2-Aminophenol 1,6-dioxygenase catalyzes ring cleavage of several ortho-aminophenols. The ring fission products spontaneously convert to picolinic acids. Resting cells of Escherichia coli DH5α/pNBZ14 harboring the genes for 2-aminophenol 1,6-dioxygenase converted 2-aminophenol and 6-amino-m-cresol to picolinic acid and 5-methylpicolinic acid with yields greater than 90%. The results provide a convenient strategy for the synthesis of substituted picolinic acids from the corresponding aminophenols. Journal of Industrial Microbiology & Biotechnology (2000) 25, 25–28.

Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by Mycobacterium Strain HL 4-NT-1

ABSTRACT 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 othero-aminophenolic compounds.

Novel organization of catechol meta pathway genes in the nitrobenzene degrader Comamonas sp. JS765 and its evolutionary implication.

The catechol meta cleavage pathway is one of the central metabolic pathways for the degradation of aromatic compounds. A novel organization of the pathway genes, different from that of classical soil microorganisms, has been observed in Sphingomonas sp HV3 and Pseudomonas sp. DJ77. In a Comamonas sp. JS765, cdoE encoding catechol 2,3-dioxygenase shares a common ancestry only with tdnC of a Pseudomonas putida strain, while codG encoding 2-hydroxymuconic semialdehyde dehydrogenase shows a higher degree of similarity to those genes in classical bacteria. Located between cdoE and cdoG are several putative genes, whose functions are unknown. These genes are not found in meta pathway operons of other microorganisms with the exception of cdoX2, which is similar to cmpX in strain HV3. Therefore, the gene cluster in JS765 reveals a third type of gene organization of the meta pathway.

Preparation of 2-aminomuconate from 2-aminophenol by coupled enzymatic dioxygenation and dehydrogenation reactions

2-Aminomuconate is an intermediate in the oxidative metabolism of tryptophan in mammals. The compound is not commercially available, and studies of its metabolism have been prevented by the lack of a chemical synthesis and the instability of the molecule. We report here the formation of 2-aminomuconate from 2-aminophenol by the coupled action of 2-aminophenol 1,6-dioxygenase and 2-aminomuconic semialdehyde dehydrogenase from Pseudomonas pseudoalcaligenes JS45, and isolation of the product by anion exchange chromatography. The overall procedure was completed within 3 h with a yield of 62%. The availability of the dicarboxyl α-amino acid provides the basis for investigation of the physiological function of 2-aminomuconate in the neuropathologically significant oxidative metabolism of tryptophan.