title: A novel meta-cleavage dioxygenase that cleaves a carboxyl-group-substituted 2-aminophenol. Purification and characterization of 4-amino-3-hydroxybenzoate 2,3-dioxygenase from Bordetella sp. strain 10d.
abstract: A bacterial strain that grew on 4-amino-3-hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4-amino-3-hydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110-fold increase in specific activity. The purified enzyme was characterized as a meta-cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4-amino-3-hydroxybenzoic acid, with the consumption of 1 mol of O2 per mol of substrate. The enzyme was therefore designated as 4-amino-3-hydroxybenzoate 2,3-dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21-kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4-amino-3-hydroxybenzoic acid. The Km and Vmax values for this substrate were 35 micro m and 12 micro mol.min-1.(mg protein)-1, respectively. Of the 2-aminophenols tested, only 4-aminoresorcinol and 6-amino-m-cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2-aminophenol 1,6-dioxygenase, in molecular mass, subunit structure and catalytic properties.
authors.affiliation: Department of Biofunctional Chemistry, Faculty of Agriculture and Division of Science of Biological Resources, Graduate School of Science and Technology, Kobe University, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takenaka
authors.forename: Tokiko
authors.initials: T
authors.lastname: Asami
authors.forename: Chika
authors.initials: C
authors.lastname: Orii
authors.forename: Shuichiro
authors.initials: S
authors.lastname: Murakami
authors.forename: Kenji
authors.initials: K
authors.lastname: Aoki
mesh_terms: D000595:Amino Acid Sequence; D001884:Bordetella / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010105:Oxygenases