A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties

Abstract

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.

A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

“…We expect that the crystal structure of S. solfataricus arylesterase will resolve the issue in the future. The result (Table 5) that the purified arylesterase/paraoxonase does not require any divalent cation, resulting from the lack of an effect of EDTA on its activity, is the same result as for the arylesterase from A. radiobacter (41) and the arylesterase/paraoxonase from the avian liver (39) but is different from that for the arylesterase from Triatoma infestans (17) (requiring Co 2ϩ and Mn 2ϩ ), the arylesterases/paraoxonases from the greenbug S. graminum (50) and mammals (27) (34,38). In conclusion, this is a novel arylesterase, displaying paraoxonase activity, from the thermoacidophilic archaeon S. solfataricus P1.…”

“…Bacterial arylesterases may be used for similar industrial and environmental purposes, especially considering the fact that they have broad substrate specificities, but little is known about their genetics and biochemistry. In addition, no arylesterase showing paraoxonase activity from archaea was reported, ex-cept for two phosphotriesterases having paraoxonase activity (34,38).…”

Crystallization and preliminary X-ray diffraction analysis of the lactonaseVmoLac fromVulcanisaeta moutnovskia

“…Recently, a protein family sharing $30% sequence identity with PTEs was identified by virtue of their ability to hydrolyze insecticides and were initially dubbed paraoxonases (Merone et al, 2005). A detailed biochemical and phylogenetic analysis later revealed that these proteins, named phosphotriesterase-like lactonases (PLLs), are in fact native lactonases endowed with promiscuous phosphotriesterase activity (Afriat et al, 2006;.…”

The Organophosphate Degradation (opd) Island-borne Esterase-induced Metabolic Diversion in Escherichia coli and Its Influence on p-Nitrophenol Degradation

“…These enzymes are believed to have evolved in soil bacteria to counter the toxic effects of OP insecticide residues released into agricultural soils (4,5). Bacterial OPH enzymes, besides showing high structural similarities with the quorumquenching lactonases, possess weak lactonase activity (6,7). Consequently the quorum-quenching lactonases are considered to be the possible progenitors of the bacterial OPH enzymes (7).…”

“…Bacterial OPH enzymes, besides showing high structural similarities with the quorumquenching lactonases, possess weak lactonase activity (6,7). Consequently the quorum-quenching lactonases are considered to be the possible progenitors of the bacterial OPH enzymes (7). Unlike the OPH enzymes, the MPHs have no structural similarity with quorum-quenching lactonases but instead are highly similar to ␤-lactamases (8).…”