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The oxygenase component of the2-aminobenzenesulfonate dioxygenase system from Alcaligenes sp. strain O-1

Department of Biology, The University, D-78457 Konstanz, Germany¹

Author for correspondence: Alasdair M. Cook. Tel: +49 7531 88 4247. Fax: +49 7531 88 2966. e-mail: alasdair.cook{at}uni-konstanz.de

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 ₂ß₂ subunit structure was deduced from the molecular masses of 134, 45 and 16 kDa for the native complex, and the and ß 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 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. K_m values of 29, 18 and 108 µM and V_max values of 140, 110 and 72 pkat for ABS, benzenesulfonate and 4-toluenesulfonate, respectively, were observed. The N-terminal amino acid sequences of the - and ß-subunits of the oxygenase component allowed PCR primers to be deduced and the DNA sequence of the -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.

Keywords: Alcaligenes sp. strain O-1, characterization of oxygenase, 2-aminobenzenesulfonate dioxygenase system, oxygenase sequence, oxygenation and deamination

Abbreviations: ABS, 2-aminobenzenesulfonate; ABSDOS, 2-ABS dioxygenase system; BS, benzenesulfonate; 3SCDO, 3-sulfocatechol dioxygenase; TS, 4-toluenesulfonate

The GenBank accession number for the sequence reported in this paper is AF109074.

^a Present address: Estación Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, E-18008 Granada, Spain.

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