A novel metaâcleavage dioxygenase that cleaves a carboxylâgroupâsubstituted 2âaminophenol: Purification and characterization of 4âaminoâ3âhydroxybenzoate 2,3âdioxygenase from Bordetella sp. strain 10d
2002
Takenaka, Shinji | Asami, Tokiko | Orii, Chika | Murakami, Shuichiro | Aoki, Kenji
A bacterial strain that grew on 4âaminoâ3âhydroxybenzoic 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 4âaminoâ3âhydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110âfold increase in specific activity. The purified enzyme was characterized as a metaâcleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4âaminoâ3âhydroxybenzoic acid, with the consumption of 1âmol of O2 per mol of substrate. The enzyme was therefore designated as 4âaminoâ3âhydroxybenzoate 2,3âdioxygenase. The molecular mass of the native enzyme was 40âkDa based on gel filtration; the enzyme is composed of two identical 21âkDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4âaminoâ3âhydroxybenzoic acid. The Km and Vmax values for this substrate were 35âµm and 12âµmol·min−1·(mgâprotein)−1, respectively. Of the 2âaminophenols tested, only 4âaminoresorcinol and 6âaminoâmâcresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2âaminophenol 1,6âdioxygenase, in molecular mass, subunit structure and catalytic properties.
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