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

• Published in: Journal of Bacteriology 1995-10, Vol.177 (20), p.5865-5871
• Main Authors: Heiss, G. (Universitat Stuttgart, Stuttgart, Germany.), Stolz, A, Kuhm, A.E, Muller, C, Klein, J, Altenbuchner, J, Knackmuss, H.J
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; BACTERIA; Bacteria - enzymology; Bacteria - genetics; Bacteriology; Base Sequence; Catechols - metabolism; Cloning, Molecular; COMPOSE ORGANOCHLORE; COMPOSE PHENOLIQUE; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; COMPUESTO ORGANICO DEL CLORO; COMPUESTOS FENOLICOS; Deoxyribonucleic acid; Dioxygenases; DNA; Escherichia coli - genetics; GENE; GENES; Genes, Bacterial; Molecular Sequence Data; Naphthalenesulfonates - metabolism; OXIDACION; Oxidation-Reduction; OXIDORREDUCTASAS; OXYDATION; OXYDOREDUCTASE; Oxygenases - genetics; Oxygenases - isolation & purification; Oxygenases - metabolism; PURIFICACION; PURIFICATION; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Salicylates - metabolism; SECUENCIA NUCLEICA; Sequence Analysis, DNA; Sequence Homology, Amino Acid; SEQUENCE NUCLEIQUE; Substrate Specificity
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/jb.177.20.5865-5871.1995

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