An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity. DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp. This is the smallest gene encoding an extradiol dioxygenase found until now. Expression of the gene in a T7 expression vector enabled purification of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-and 4-chlorocatechol, and 3-and 4-methylcatechol. Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.A bacterial strain (strain BN6) which is able to degrade amino-and hydroxynaphthalenesulfonates is currently being studied in this laboratory. This strain oxidizes substituted naphthalenesulfonates to the corresponding substituted 1,2-dihydroxynaphthalenes by a reaction catalyzed by a desulfonating dioxygenase. Subsequent metabolism of the substituted 1,2-dihydroxynaphthalenes (1,2-DHNs) to substituted salicylates follows the common naphthalene degradative pathway. Strain BN6 does not mineralize naphthalenesulfonates, since the salicylates are not further oxidized (24,25,33,34). Cloning the genes for the metabolism of these naphthalenesulfonates would facilitate mobilization into salicylate-degrading bacterial strains, thereby accomplishing the complete degradation of naphthalenesulfonates in a single strain. Previously, the 1,2-DHN dioxygenase (DHNDO), which is part of this metabolic pathway, was purified to homogeneity and biochemically characterized. The enzyme oxidized 1,2-DHN, 2,3-dihydroxybiphenyl (2,3-DHBP), and some other aromatic diols (25). In the present study, we attempted to clone the corresponding gene from strain BN6. Although we obtained clones derived from strain BN6 which were able to oxidize 2,3-DHBP, the encoded gene was unexpectedly not involved in the degradation of naphthalenesulfonates. The extradiol dioxygenase (DO) encoded by this clone was examined in the present study. MATERIALS AND METHODSBacterial strains and culture conditions. The isolation and characterization of strain BN6 have been described previously (33). The strain has been deposited at the Deutsche Sammlung von Mikroorganismen, Brunswick, Germany, as DSM 6383. For the isolation of genomic DNA, the strain was grown in nutrient broth. Escherichia coli DH5␣ was the host for construction of the genomic library. E. coli JM 109 was used for subcloning and isolation of DNA for sequencing. Fo...

Two hydrogenation reactions in the initial steps of degradation of 2,4,6-trinitrophenol produce the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. The npdH gene (contained in the npd gene cluster of the 2,4,6-trinitrophenol-degrading strain Rhodococcus opacus HL PM-1) was shown here to encode a tautomerase, catalyzing a proton shift between the aci-nitro and the nitro forms of the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. An enzyme (which eliminated nitrite from the aci-nitro form but not the nitro form of the dihydride complex of 2,4,6-trinitrophenol) was purified from the 2,4,6-trinitrophenol-degrading strain Nocardioides simplex FJ2-1A. The product of nitrite release was the hydride Meisenheimer complex of 2,4-dinitrophenol, which was hydrogenated to the dihydride Meisenheimer complex of 2,4-dinitrophenol by the hydride transferase I and the NADPH-dependent F 420 reductase from strain HL PM-1. At pH 7.5, the dihydride complex of 2,4-dinitrophenol is protonated to 2,4-dinitrocyclohexanone. A hydrolase was purified from strain FJ2-1A and shown to cleave 2,4-dinitrocyclohexanone hydrolytically to 4,6-dinitrohexanoate.2,4,6-Trinitrophenol (TNP) and 2,4-dinitrophenol (DNP) are nitroaromatic compounds of versatile use in chemical synthesis. They occur as off-stream chemicals during the production of aniline, which is one of the most important starting materials in chemical synthesis. Furthermore, TNP and its salts have been used as explosives. Large quantities of TNP in waste streams of aniline production necessitate remediation. Several bacteria of the Actinomycetales family (notably of the genera Rhodococcus and Nocardioides) grow aerobically on TNP and/or DNP and utilize the compounds as sole nitrogen, carbon, and energy sources (3,4,10,19). This capacity can be harnessed for bioremediation once we understand the underlying mechanisms of these processes.It was previously established that two hydrogenations take place in the initial attack on TNP (6,7,8,11) (Fig. 1A, panel 1). In Rhodococcus opacus HL PM-1, TNP is hydrogenated at the aromatic nucleus by the hydride transferase II (HTII) encoded by npdI (Fig. 1B) and the NADPH-dependent F 420 reductase (NDFR) encoded by npdG. The hydride Meisenheimer complex of TNP (H Ϫ -TNP) (Fig. 1A, panel 2) thereby formed is further hydrogenated by the hydride transferase I (HTI) encoded by npdC and the NDFR, producing the dihydride Meisenheimer complex of TNP (2H Ϫ -TNP) (panel 3a). In Nocardioides simplex FJ2-1A, the same reactions take place except that a single hydride transferase performs both hydrogenations (7).More than a decade ago, 4,6-dinitrohexanoate was identified as a dead-end metabolite of TNP degradation resulting from two hydride transfers to TNP. This observation coincided with the hypothesis that 2HϪ -TNP was a dead-end metabolite of TNP degradation (11). Much later it was suggested that nitrite is eliminated from 2H Ϫ -TNP to produce the hydride Meisen-Ϫ -TNP is a metabolite of productive TNP degradation. Detection of H Ϫ -DNP suggest...

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