Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids

Abstract

1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2" for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobiis Ql. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3-and 3,4-dihydroxybiphenyl, catechol, and 3-methyl-and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalenedegrading bacteria.Amino-and hydroxynaphthalenesulfonic acids (ANS and HNS, respectively) serve as building blocks for the largescale synthesis of azo dyes. Since arylsulfonates are very rare among natural compounds (21), naphthalenesulfonic acids are referred to as xenobiotics. Arylsulfonates are major pollutants of the environment, and the contamination of the Rhine River by sulfur-organic compounds is largely due to this class of compounds (25). Nevertheless, bacteria which degrade naphthalenesulfonic acids have repeatedly been isolated (4, 5, 29-31, 41, 43). Brilon et al. (4, 5) selected pseudomonads which degraded naphthalene-2-sulfonic acid (2NS) and naphthalene-1-sulfonic acid (1NS). The initial reaction in the degradation of iNS and 2NS is catalyzed by a 1,2-dioxygenase which weakens the carbon-sulfur bond.

Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions

“…Other researchers (3,7,34,52) M-1 cm1-, 2% is 1,450 M-1 cm-1, 6323 is 2,520 M1 cm-', and e is 1,890 M-1 cm-1. was dissolved in D20 and then NaOH was added.…”

“…Misidentification of tHBPA as the cis isomer in previous work (3,7,34,52) has been a factor which has complicated a complete understanding of the naphthalene pathway. Barnsley (3) identified the first product of ring cleavage of 1,2-dihydroxynaphthalene as HCCA and showed that this compound isomerized spontaneously to form an equilibrium mixture with a compound that he identified as cHBPA.…”

Identification and functional analysis of the genes for naphthalenesulfonate catabolism by Sphingomonas xenophaga BN6

“…In silico identification of the genes encoding the NsaC, NsaD, NsaE and NsaF involved in the degradation of naphthalenesulfonates NsaC and NsaE from S. xenophaga BN6 have previously been purified and their N-terminal amino acid sequences determined (Kuhm et al, 1991a(Kuhm et al, , 1992. This allowed the identification of ORF7 and ORF13 as the genes encoding the NsaC and NsaE of the naphthalenesulfonate pathway, because the N-terminal 29 or 26 aa of the predicted protein sequences were identical to the data obtained experimentally by protein sequencing.…”

“…Three of the relevant enzymes, NsaC, NsaD and NsaE, have been purified and biochemically characterized. Furthermore, the N-terminal amino acid sequences of NsaC and NsaE have been determined (Kuhm et al, 1991a(Kuhm et al, , 1992(Kuhm et al, , 1993.…”

“…NsaE and NsaD were assayed spectrophotometrically, as described by Kuhm et al (1992Kuhm et al ( , 1993.…”