The oxygenase component of the 2-aminobenzenesulfonate dioxygenase system from Alcaligenes sp strain O-1

Growth of Alcaligenes sp, strain O-1 with 2-aminobenzenesulfonate (ABS; ort hanilate) as sole source of carbon and energy requires expression of the so luble, multicomponent 2-aminobenzenesulfonate 2,3-dioxygenase system (deami nating) (ABSDOS) which is plasmid-encoded. ABSDOS was separated by anion-ex change chromatography to yield a flavin-dependent reductase component and a n iron-dependent oxygenase component. The oxygenase component was purified to about 98% homogeneity and an alpha(2)beta(2) subunit structure was deduc ed from the molecular masses of 134, 45 and 16 kDa for the native complex, and the alpha and beta subunits, respectively. Analysis of the amount of ac id labile sulfur and total iron, and the UV spectrum of the purified oxygen ase component indicated one [2Fe-2S] Rieske centre per ct subunit, The inhi bition of activity by the iron-specific chelator o-phenanthroline indicated the presence of an additional iron-binding site. Recovery of active protei n required strictly anoxic conditions during all purification steps. The FA D-containing reductase could not be purified. ABSDOS oxygenated nine sulfon ated compounds; no oxygen uptake was detected with carboxylated aromatic co mpounds or with aliphatic sulfonated compounds. K-m values of 29, 18 and 10 8 mu M and V-max values of 140, 110 and 72 pkat for ABS, benrenesulfonate a nd 4-toluenesulfonate, respectively, were observed, The N-terminal amino ac id sequences of the alpha- and beta-subunits of the oxygenase component all owed PCR primers to be deduced and the DNA sequence of the alpha-subunit wa s thereafter determined. Both redox centres were detected in the deduced am ino acid sequence. Sequence data and biochemical properties of the enzyme s ystem indicate a novel member of the class IB ring-hydroxylating dioxygenas es.