Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.

Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization--time of flight--mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD(50) dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose.

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