Sequence analysis and initial characterization of two isozymes of hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45.

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 degrees C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60 degrees C. HAB mutase activity in extracts of P. pseudoalcaligenes JS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and the habB 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 tuberculosis H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. coli containing 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 of habB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.

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.

Oxidation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5.

Previous studies demonstrated that Alcaligenes eutrophus A5 transforms 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) to 4-chlorobenzoate via a meta-ring fission product. The initial reactions could be catalyzed by either monooxygenase or dioxygenase enzymes. In the present study, a transient intermediate that accumulated during the transformation of DDT by the biphenyl-grown cells was identified as 1,1,1-trichloro-2-(4-chlorophenyl-2,3-dihydro-4,6-cyclohexadiene)-2-(4'- chlorophenyl)ethane (DDT-2,3-dihydrodiol) on the basis of mass spectral analysis after n-butylboronic acid derivatization. The dihydrodiol undergoes a characteristic acid-catalyzed dehydration to produce phenols. 1H-NMR indicated a cis-relative stereochemistry. The results indicate that the biphenyl dioxygenase from A. eutrophus A5 catalyzes the dihydroxylation of DDT at the unsubstituted carbons on the aromatic ring to produce DDT-2,3-dihydrodiol.

Bacterial degradation of m-nitrobenzoic acid.

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.

Aerobic degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5.

Biotransformation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5 was demonstrated by analysis of ethyl acetate-extracted products from resting cell cultures. Gas chromatography-mass spectrometry characterization of the neutral extracts revealed two hydroxy-DDT intermediates (m/z = 370) with retention times at 19.55 and 19.80 min that shared identical mass spectra. This result suggested that the hydroxylations occurred at the ortho and meta positions on the aromatic ring. UV-visible spectrum spectrophotometric analysis of a yellow metabolite in the culture supernatant showed a maximum A402 with, under acidic and basic conditions, spectrophotometric characteristics similar to those of the aromatic ring meta-cleavage products. 4-Chlorobenzoic acid was detected by thin-layer chromatography radiochemical scanning in samples from mineralization experiments by comparison of Rf values of [14C]DDT intermediates with that of an authentic standard. These results were further confirmed by gas chromatography-mass spectrometry analysis. This study indicates that DDT appears to be oxidized by a dioxygenase in A. eutrophus A5 and that the products of this oxidation are subsequently subjected to ring fission to eventually yield 4-chlorobenzoic acid as a major stable intermediate.