Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6

Abstract

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...

Molecular Basis for the Stabilization and Inhibition of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by t-Butanol

“…The reversible substrate inhibition observed in DHBD has been reported for a number of other DHB-cleaving extradiol dioxygenases (13,(35)(36)(37)(38) as well as for a 2,3-dihydroxyphenyl propionate cleaving enzyme (39) although it has only rarely been reported for catechol 2,3-dioxygenases (40). However, for the other DHB cleaving enzymes, it is not clear what proportion of the decrease in the initial rate of DHB cleavage at high concentrations of DHB is due to reversible substrate inhibition and irreversible suicide inhibition, respectively.…”

“…However, for the other DHB cleaving enzymes, it is not clear what proportion of the decrease in the initial rate of DHB cleavage at high concentrations of DHB is due to reversible substrate inhibition and irreversible suicide inhibition, respectively. Notably, the initial rates of cleavage of substituted catechols by a DHBD of strain BN6 could not be fitted to substrate inhibition Equation 2 (38). DHBD of B. cepacia LB400 is clearly subject to both modes of inhibition by both DHB and 3-ethylcatechol.…”

Expression, Purification, and Characterization of 2′-Aminobiphenyl-2,3-diol 1,2-dioxygenase from Carbazole-degraderPseudomonas resinovoransStrain CA10

“…This strict preference for substrates having a biphenyl moiety was found for meta-cleavage enzymes involved in the biphenyl or dibenzofuran degradation pathway of an other class (class II). 20,[30][31][32][33] CarB from strain CA10 was originally isolated as a meta-cleavage enzyme for 2?-aminobiphenyl-2,3-diol involved in carbazole degradation, 9) but substitution at the 2?-position of 2,3-dihydroxybiphenyl decreased the a‹nity of the enzyme for the substrates. The Km values of DbfB, a meta-cleavage enzyme involved in dibenzofuran degradation by strain RW1, are 8.5 mM for 2,3-dihydroxybiphenyl and 11 mM for 2,2?,3-trihydroxybiphenyl.…”

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls(PCBs).

“…The third enzyme is a 2,3-dihydroxybiphenyl dioxygenase that is involved in the ring meta-cleavage at the 1,2 position (Eltis et al, 1993;Heiss et al, 1995;Khan et al, 1996). The enzyme from P. pseudoalcaligenes KF707 was associated as a homooctamer .…”