CHARACTERIZATION OF A 2,3-DIHYDROXYBIPHENYL DIOXYGENASE FROM THE NAPHTHALENESULFONATE-DEGRADING BACTERIUM STRAIN BN6

0An extradiol dioxygenase was cloned from the naphthalenesulfonate-deg rading 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. Thi s is the smallest gene encoding an extradiol dioxygenase found until n ow. Expression of the gene in a T7 expression vector enabled purificat ion of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacryl amide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybip henyl, 3-isopropylcatechol, 3- and 4-chlorocatechol, and 3- and 4-meth ylcatechol. Since the ability to convert 3-chlorocatechol is an unusua l characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid seq uence differed from the corresponding sequence of the 1,2-dihydroxynap hthalene dioxygenase, which had been determined earlier from the enzym e purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.