Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase.
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.
Merone
Luigia
L
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.
Mandrich
Luigi
L
Porzio
Elena
E
Rossi
Mosé
M
Müller
Susanne
S
Reiter
Georg
G
Worek
Franz
F
Manco
Giuseppe
G
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
D002619:Chemical Warfare Agents; D008024:Ligands; D012967:Sodium Dodecyl Sulfate; D002265:Carboxylic Ester Hydrolases; D044345:Phosphoric Triester Hydrolases