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Reversed Enantioselectivity of DFPase by Rational Design

Handedness of enantiomers“Handedness” of enantiomers with amino
acids as examples (Source: Wikipedia/NASA)

The enzyme Diisopropyl fluorophosphatase (DFPase) catalyses the hydrolysis of the toxic organophosphorus compound Diisopropyl fluorophosphat (DFP) and a range of highly toxic nerve agents of the so called G-series. This class of nerve agents includes compounds like tabun (GA), sarin, (GB), soamn (GD) and cyclosarin (GF). DFP does not contain any stereocenters and is achiral but all mentioned G-type nerve agents have four different substitutents at the central phosphorus atom. Therefore this phosphorus atom is asymmetric and forms a stereocenter. GA, GB, GF are chiral and exist as pair of enantiomers, which relate to each other like left hand and right hand (GD is a more complicated case as there is an additional stereocenter in one of the side chains).

There is no difference between the enantiomers in chemical reactions that take place in an achiral environment. An example for this is the aqueous hydrolysis of the agents at high pH. But when interacting with other chiral molecules like proteins they diplay distinct differences. The main “target” for the nerve agents is the enzyme acteylcholinesterase (AChE), which is inhibited by the nerve agents by the formation of a stable covalent adduct. One of the two enantiomers shows a significantly higher inhibitory power compared to the other, which is only mildly inhibiting or even non-inhibiting. A similar effect is seen when hydrolyzing (and detoxifying) the agents using DFPase as an enzymatic catalyst. Wildtype DFPase prefers the less toxic enantiomer. This is especially problematic for medical application (in vivo or topical) as rapid reduction of toxicity is of special importance. A potential strategy to reverse enantioselectivity of DFPase is to alter the active site of the enzyme by mutagenesis.

Model of the phosphoenzyme intermediate of DFPase for the substrate sarinModel of the phosphoenzyme intermediate
of DFPase for the substrate sarin

Due to the legal restrictions for handling highly toxic compounds* the standard approach for mutagenesis using evolutionary methods and high-throuput screening of mutant libraries could not be used. Instead we turned to rational protein design. One should note that reversing enantioselectivity of an enzyme (and keeping activity) is not a trivial task and the number of publications dealing with this issues is still limited. Detailed knowledge about the DFPase structure and the reaction mechanism, which is though to proceed via a phosphoenzyme intermediate, formed an invaluable base for planning the mutations.

The constructed mutants finally displayed the desired reversed enantioselectivity without loosing enzymatic activity. The mutants rather showed enhanced activities (up to 8-fold higher in case of GB). For an optimal combination of selectivity , activity and substrate affinity the mutants should be mixed with wildtype DFPase. Using such mixtures total hydrolysis of the agents can be achieved 4-fold faster even at low substrate concentrations (where substrate affinity is an issue) with an even faster reduction in toxicity as in the first phase of the reaction the more toxic enantiomer is hydrolyzed faster.

Structures of the mutants were deposited in the PDB under accession codes 3HLI and 3HLH.

* Work with nerve agents was conducted in accordance with the Chemical Weapons Convention (CWC) at the Bundeswehr Institute for Pharmacology and Toxicology,

Reversed Enantioselectivity of Diisopropyl Fluorophosphatase against Organophosphorus Nerve Agents by Rational Design.
Melzer M, Chen JC, Heidenreich A, Gäb J, Koller M, Kehe K, Blum MM.
J. Am. Chem. Soc. 2009;131(47):17226-17232.

Research Highlight in Nature Chemistry:

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.


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