A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: purification, characterization, and expression.
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 (C₄) to laurate (C₁₂). The k(cat)/K(m) ratios for trans-dienelactone and p-nitrophenyl caprylate (C₈), the best substrate, were 92.5 and 54.7 s⁻¹ μM⁻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.


CONCLUSIONS
The enzyme is the first characterized archaeal dienelactone hydrolase.
Park
Young-Jun
YJ
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Korea.
Yoon
Sung-Jin
SJ
Lee
Hee-Bong
HB
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C046130:carboxymethylenebutenolidase