Purification and characterization of a novel l-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli

  • Isamu Tashima
  • Takashi Yoshida
  • Yoshihiro Asada
  • Tetsuo Ohmachi
Biotechnologically Relevant Enzymes and Proteins

Abstract

l-2-Amino-Δ2-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu2+ and Mn2+ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of Km (0.35 mM) and Vmax (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine.

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Isamu Tashima
    • 1
  • Takashi Yoshida
    • 1
  • Yoshihiro Asada
    • 1
  • Tetsuo Ohmachi
    • 1
  1. 1.Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life ScienceHirosaki UniversityHirosakiJapan

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