DFPase

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Introduction

Diisopropyl fluorophosphatase (DFPase; EC 3.1.8.2) from the squid Loligo vulgaris is a 35 kDa protein consisting of 314 amino acids. It is an effective Ca²⁺-dependent phosphotriesterase that can hydrolyze a series of Organophosphorus compounds that as irreversible acetylcholinesterase (AChE) inhibitors. Its substrates include diisopropyl fluorophosphate (DFP) and a series of highly toxic G-type organophosphorus (OP) nerve agents, such as sarin (GB), tabun (GA), and so on.

Figure 1. Structures of the DFPase substrates DFP, GB, GF, GD and GA. Stereocentres are indicated by asterisks (Chen, J.C.; et al. 2010)

The hydrolysis reaction catalyzed by DFPase forms phosphate or phosphonate and fluoride ions, thereby detoxifying organophosphorus agents. Since the enzyme can be expressed in large quantities, and shows high tolerance to organic solvents and strong stability, DFPase is considered a promising candidate for enzymatic decontamination. The His287 near the active site observed in the DFPase structure obtained for the first time is presumed to be the general base in the reaction. However, subsequent mutation experiments on His287 showed that even if replaced by aliphatic residues (such as leucine), the overall activity of the enzyme hardly changed. A series of subsequent researches studied the function and structure of the DFPase mechanism, including mutants of active-site residues, isotope labeling, and the 2.2 Å neutron structure of DFPase. This proposed a reaction mechanism involving direct nucleophilic attack by Asp229 and phosphoenzyme intermediate.

Figure 2. Potential reaction mechanisms for DFPase (Chen, J.C.; et al. 2010)

Structure and active-site environment

Many DFPase structures have been reported. In addition to holoenzymes, several structures of active-site mutants and the structure of complex with the designed substrate analogue DcPPA have also been resolved. It can be seen from the overall structure that the protein adopts a six-blade β-propeller surrounding the central water tunnel and coordinates two calcium ions. The high-affinity calcium Ca1 located in the central water tunnel is necessary to maintain structural integrity and shows rare Ca-N coordination by the side chain of His274. The low-affinity calcium ion Ca2 is located at the bottom of the active site pocket, which is essential for the catalytic activity of the enzyme. Calcium ions are further coordinated below the active site by two water molecules linked to the central water tunnel and a solvent molecule (W33) in the active site pocket.

The catalytic calcium coordination residues have undergone extensive mutations, and the changes in these calcium coordination residues have important impact on binding and activity. Among the 10 mutants created, only N175D and N120D showed extremely weak enzymatic activity against DFP, while the remaining 8 mutants were inactive. When there is only one negative charge left in the active site, the inactivity of these mutants can be attributed to the loss of catalytic calcium. Glu21 seems to play a vital role in calcium coordination, because once Glu21 is mutated, no matter how much negative charge there is nearby, it will cause the loss of calcium binding function.

Figure 3. Fold of DFPase and catalytic calcium environment (Chen, J.C.; et al. 2010)

Re-engineering DFPase

DFPase neutron structure and isotope labeling studies support the nucleophilic attack of Asp229 on the P atom of the substrate, leading to the formation of phosphoenzyme intermediates and subsequent hydrolysis. This information can play important roles in rational design to improve the overall activity and detoxification properties of the enzyme. In addition, the structure of DFPase highlights the rigidity of the active site environment, and the co-crystal structure with the substrate analog DcPPA has little conformational change in the active site after binding, making DFPase an example of a lock-and-key enzyme. Wild-type DFPase preferentially binds and hydrolyzes the less toxic stereoisomers of G type nerve agents. For these re-engineering, a phosphatase intermediate models were generated for the R_P and S_P stereoisomers of cyclosalin. The active site has been modified to accommodate the more toxic R_P stereoisomer. By introducing shorter side chains at residues 37, 144, and 146, a more open binding pocket is created on one side of the active site, and on the opposite face, it is restricted by introducing a larger side chain at residue 195. The two mutants obtained were E37A/Y144A/R146A/T195M and E37D/Y144A/R146A/T195M. The activity of these two mutants was tested and their X-ray structures were also analyzed. Both showed an overall enhanced activity against racemic mixtures of cyclosarin (GF) and sarin (GB). Compared with wild-type DFPase, the stereoselectivity of mutant E37A/Y144A/R146A/T195M to the S_P stereoisomer of cyclosarin (GF) is nearly 200 times higher. The mutants have higher specific activity on DFP, GF and GB, and with the rapid hydrolysis of SP stereoisomers, the detoxification properties are significantly enhanced.