Purification andProperties ofan Organophosphorus AcidAnhydrase froma Halophilic Bacterial Isolate

A moderately halophilic bacterial isolate hasbeenfoundtopossesshighlevels ofenzymatic activity against several highly toxic organophosphorus compounds. Thepredominant enzyme,designated organophosphorus acidanhydrase 2,hasbeenpurified 1,000-fold tohomogeneity andcharacterized. Theenzyme isa single polypeptide with amolecular weight of60,000. Withdiisopropylfluorophosphate asasubstrate, theenzymehas optimumactivity atpH8.5and50°C, anditisstimulated bymanganeseandcobalt. Organophosphorus acid(OPA)anhydrases are enzymes that arecapable ofcatalytically hydrolyzing awidevariety of organophosphorus cholinesterase inhibitors, among them diisopropylfluorophosphate (DFP),thechemical warfare agentssoman (0-1,2,2-trimethylpropyl methylphosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), andtabun(ethyl N,N-dimethylphosphoramidocyanidate), andthepesticides parathion (diethyl p-nitrophenyl phosphorothioate) andparaoxon (diethyl p-nitrophenyl phosphate) (10,12,13). Enzymessuchasthese areofinterest fortheir potential use indecontamination anddemilitarization of these extremely toxic materials. Inthepast,these enzymes were knownvariously as DFPases, somanases,parathion hydrolases, or paraoxonases,depending on theassaysubstrate used.Sources ofthese enzymes rangefrombacteria andprotozoans tohigher mammals,including humans, and thenumberofenzymes foundhasgreatly increased inrecent years(10). Theproliferation ofbothenzymes andenzyme names ledtotheadoption ofthename OPA anhydrase during theFirst DFPaseWorkshop(Marine Biological Laboratory, WoodsHole,Mass., June1987) todescribe these related enzymes.Itwas planned thatthis name beuseduntil thenatural substrates andfunctions ofthese enzyme are identified. Preliminary studies ofOPA anhydrases from various sourceshavedemonstrated that these enzymes differ insubstrate specificity, sensitivity toinhibitors, activation bymetals, andmolecular weight (10). Purification andcharacterization ofthese enzymes, suchastheone described in this report, may assist inthedetermination ofthetruenature oftheir substrates, specificity, andmolecular structure. Thesourceoftheenzyme tobediscussed istheobligately halophilic bacterial isolate designated JD6.5,whichwas isolated froma warm salt spring. Thisisolate was foundto possesshighlevels ofDFP-hydrolyzing OPA anhydrase activity (3). Inthis report we describe thepurification and characterization ofOPA anhydrase 2 (OPAA-2), thepredominant enzyme fromJD6.5.