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; orthanilate) as sole source of carbon and energy requires expression of the soluble, multicomponent 2-aminobenzenesulfonate 2,3-dioxygenase system (deaminating) (ABSDOS) which is plasmid-encoded. ABSDOS was separated by anion-exchange chromatography to yield a flavin-dependent reductase component and an iron-dependent oxygenase component. The oxygenase component was purified to about 98% homogeneity and an alpha2beta2 subunit structure was deduced 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 acid labile sulfur and total iron, and the UV spectrum of the purified oxygenase component indicated one [2Fe-2S] Rieske centre per alpha subunit. The inhibition of activity by the iron-specific chelator o-phenanthroline indicated the presence of an additional iron-binding site. Recovery of active protein required strictly anoxic conditions during all purification steps. The FAD-containing reductase could not be purified. ABSDOS oxygenated nine sulfonated compounds; no oxygen uptake was detected with carboxylated aromatic compounds or with aliphatic sulfonated compounds. Km values of 29, 18 and 108 microM and Vmax values of 140, 110 and 72 pkat for ABS, benzenesulfonate and 4-toluenesulfonate, respectively, were observed. The N-terminal amino acid sequences of the alpha- and beta-subunits of the oxygenase component allowed PCR primers to be deduced and the DNA sequence of the alpha-subunit was thereafter determined. Both redox centres were detected in the deduced amino acid sequence. Sequence data and biochemical properties of the enzyme system indicate a novel member of the class IB ring-hydroxylating dioxygenases.
Mampel
Jörg
J
Department of Biology, The University, D-78457 Konstanz, Germany1.
Ruff
Jürgen
J
Department of Biology, The University, D-78457 Konstanz, Germany1.
Junker
Frank
F
Department of Biology, The University, D-78457 Konstanz, Germany1.
Cook
Alasdair M
AM
Department of Biology, The University, D-78457 Konstanz, Germany1.
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D006899:Mixed Function Oxygenases / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D013425:Sulfanilic Acids / Q000378:metabolism
D013425:Sulfanilic Acids; D006899:Mixed Function Oxygenases; C402707:2-aminobenzenesulfonate 2,3-dioxygenase (deaminating); C100048:2-sulfanilic acid