title: Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase.
abstract: The thermostable Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus (SsoPox) hydrolyzes lactones and, at a lower rate, neurotoxic organophosphorus compounds. The persistent demand of detoxification tools in the field of agricultural wastes and restoring of conditions after terrorist acts prompted us to exploit SsoPox as a "starter" to evolve its ancillary nerve agents hydrolytic capability. A directed evolution strategy yielded, among several variants, the single mutant W263F with k(cat) and specificity constant against paraoxon 16- and 6-fold enhanced, respectively, compared to the wild type. Furthermore, a phenomenon of enzyme activation by SDS has been observed, which allowed to increase those values 150- and 28-fold, respectively. The activity of SsoPox against the deadly nerve gas Cyclosarin has been reported for the first time and proved to be substantially unaffected for variant W263F. Finally, outperforming efficiency of W263F was demonstrated, under severe stressing conditions, with respect to the best known phosphotriesterase PTE from Brevundimonas diminuta.
authors.affiliation: Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.
authors.forename: Luigia
authors.initials: L
authors.lastname: Merone
authors.forename: Luigi
authors.initials: L
authors.lastname: Mandrich
authors.forename: Elena
authors.initials: E
authors.lastname: Porzio
authors.forename: Mosé
authors.initials: M
authors.lastname: Rossi
authors.forename: Susanne
authors.initials: S
authors.lastname: Müller
authors.forename: Georg
authors.initials: G
authors.lastname: Reiter
authors.forename: Franz
authors.initials: F
authors.lastname: Worek
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Manco
mesh_terms: D001673:Biodegradation, Environmental / Q000187:drug effects; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002619:Chemical Warfare Agents / Q000378:metabolism*; D019020:Directed Molecular Evolution; D057075:Enzyme Assays; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D009154:Mutation / Q000235:genetics; D044345:Phosphoric Triester Hydrolases / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D016945:Sulfolobus acidocaldarius / Q000187:drug effects / Q000201:enzymology*; D048229:Sulfolobus solfataricus / Q000187:drug effects / Q000201:enzymology*; D013696:Temperature
chemical_list: D002619:Chemical Warfare Agents; D008024:Ligands; D012967:Sodium Dodecyl Sulfate; D002265:Carboxylic Ester Hydrolases; D044345:Phosphoric Triester Hydrolases