A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties.
A new gene from the hyperthermophilic archaeon Sulfolobus solfataricus MT4, coding for a putative protein reported to show sequence identity with the phosphotriesterase-related protein family (PHP), was cloned by means of the polymerase chain reaction from the S. solfataricus genomic DNA. In order to analyse the biochemical properties of the protein an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble form at 5 mg/l of E. coli culture, was purified to homogeneity and characterized. In contrast with its mesophilic E. coli counterpart that was devoid of any tested activity, the S. solfataricus enzyme was demonstrated to have a low paraoxonase activity. This activity was dependent from metal cations with Co(2+), Mg(2+) and Ni(2+) being the most effective and was thermophilic and thermostable. The enzyme was inactivated with EDTA and o-phenantroline. A reported inhibitor for Pseudomonas putida phosphotriesterase (PTE) had no effect on the S. solfataricus paraoxonase. The importance of a stable paraoxonase for detoxification of chemical warfare agents and agricultural pesticides will be discussed.
Merone
Luigia
L
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino, Napoli, Italy.
Mandrich
Luigi
L
Rossi
Mosè
M
Manco
Giuseppe
G
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D044345:Phosphoric Triester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D001051:Apoenzymes; D044345:Phosphoric Triester Hydrolases