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Purification and Characterization of the Arylsulfatase Synthesized by Pseudomonas aeruginosa PAO During Growth in Sulfate-Free Medium and Cloning of the Arylsulfatase Gene (atsA)

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1995

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Beil, Stefan
Kehrli, Hans
James, Peter
Staudenmann, Werner
Leisinger, Thomas
Kertesz, Michael A.

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Published

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European Journal of Biochemistry. 1995, 229(2), pp. 385-394. ISSN 0014-2956. eISSN 1432-1033. Available under: doi: 10.1111/j.1432-1033.1995.0385k.x

Zusammenfassung

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PAO1 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDS/PAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57°C and pH 8.9, and a Km of 105 myM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in P. aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5 751 mutant of P. aeruginosa PAO1. Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30 40% and 25 30% identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the P. aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15 17' on the P. aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

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Fachgebiet (DDC)
570 Biowissenschaften, Biologie

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Arylsulfatase, Pseudomonas aeruginosa, Sulfur metabolism, Sulfate limitation

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ISO 690BEIL, Stefan, Hans KEHRLI, Peter JAMES, Werner STAUDENMANN, Alasdair M. COOK, Thomas LEISINGER, Michael A. KERTESZ, 1995. Purification and Characterization of the Arylsulfatase Synthesized by Pseudomonas aeruginosa PAO During Growth in Sulfate-Free Medium and Cloning of the Arylsulfatase Gene (atsA). In: European Journal of Biochemistry. 1995, 229(2), pp. 385-394. ISSN 0014-2956. eISSN 1432-1033. Available under: doi: 10.1111/j.1432-1033.1995.0385k.x
BibTex
@article{Beil1995Purif-6698,
  year={1995},
  doi={10.1111/j.1432-1033.1995.0385k.x},
  title={Purification and Characterization of the Arylsulfatase Synthesized by Pseudomonas aeruginosa PAO During Growth in Sulfate-Free Medium and Cloning of the Arylsulfatase Gene (atsA)},
  number={2},
  volume={229},
  issn={0014-2956},
  journal={European Journal of Biochemistry},
  pages={385--394},
  author={Beil, Stefan and Kehrli, Hans and James, Peter and Staudenmann, Werner and Cook, Alasdair M. and Leisinger, Thomas and Kertesz, Michael A.}
}
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