Park_2010_Biochim.Biophys.Acta_1800_1164

BACKGROUND: Dienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli. METHODS: The enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene. RESULTS: The enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 degrees C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 degrees C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C(4)) to laurate (C(1)(2)). The k(cat)/K(m) ratios for trans-dienelactone and p-nitrophenyl caprylate (C(8)), the best substrate, were 92.5 and 54.7 s(-)(1) muM(-)(1), respectively. CONCLUSIONS: The enzyme is a typical dienelactone hydrolase belonging to alpha/beta hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site. GENERAL SIGNIFICANCE: The enzyme is the first characterized archaeal dienelactone hydrolase.