Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313.
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 alpha-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C(4) to C(12), required oxygen and Fe(2+) 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 alpha-ketoglutarate-dependent dioxygenases, AtsK accepted a range of alpha-keto acids as co-substrates, including alpha-ketoglutarate (K(m) 140 microm), alpha-ketoadipate, alpha-ketovalerate, and alpha-ketooctanoate. The measured K(m) values for hexyl sulfate and SDS were 40 and 34 microm, respectively. The apparent M(r) of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric. The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the alpha-ketoglutarate-dependent dioxygenase family.
Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland.
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D000447:Aldehydes / Q000378:metabolism; D000476:Alkanesulfonates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D016958:Pseudomonas putida / Q000201:enzymology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013386:Succinates / Q000378:metabolism; D013429:Sulfatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism
D000447:Aldehydes; D000476:Alkanesulfonates; D007656:Ketoglutaric Acids; D011994:Recombinant Proteins; D013386:Succinates; D013431:Sulfates; C010463:n-hexanal; D007501:Iron; D010105:Oxygenases; D013429:Sulfatases; C019244:alkylsulfatase; D001205:Ascorbic Acid; D010100:Oxygen