Purification and characterization of a novel L-2-amino-Delta2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli.

L-2-Amino-Delta2-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 degrees 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.