A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: Purification,characterization, and expression |
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Authors: | Young-Jun Park Sung-Jin Yoon Hee-Bong Lee |
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Institution: | Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, 200-701, Korea |
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Abstract: | BackgroundDienelactone 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.MethodsThe 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.ResultsThe enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °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 °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 kcat/Km ratios for trans-dienelactone and p-nitrophenyl caprylate (C8), the best substrate, were 92.5 and 54.7 s−1 μM−1, respectively.ConclusionsThe enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site.General significanceThe enzyme is the first characterized archaeal dienelactone hydrolase. |
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Keywords: | IPTG isopropyl-β-d-thiogalactopyranoside SDS sodium dodecyl sulfate PAGE polyacrylamide gel electrophoresis HPLC high pressure liquid chromatography CHAPS 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate PNP p-nitrophenyl α-NA α-naphthyl acetate PLP pyridoxal 5'-phosphate PGO phenylglyoxal PCMB p-chloromercuribenzoate DPC diethyl pyrocarbonate DFP diisopropylfluorophosphate PMSF phenylmethylsulfonyl fluoride EDTA ethylene diaminetetraacetic acid MALDI-TOF-MS matrix-assisted laser-desorption ionization time-of-flight mass spectrometry PCR polymerase chain reaction |
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