Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313

Abstract

The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the α-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C4 to C12, required oxygen and Fe2+ for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized α-ketoglutarate-dependent dioxygenases, AtsK accepted a range of α-keto acids as eo-substrates, including α-ketoglutarate (Km 140 pM), α-ketoadipate, α-ketovalerate, and α-ketooctanoate. The measured Km values for hexyl sulfate and SDS were 40 and 34 pM, respectively. The apparent My of the purified enzyme of 121,000 was consistent with a homotetramerie structure, which is unusual for this enzyme superfamily, members of which are usually mo-nomerie or dimerie. The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytie alkylsulfatase and a novel member of the α-ketoglutarate-dependent dioxygenase family.