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Early history of the squid DFPase

Tertiary structure of DFPase with the two calcium ions shown as green spheres

Drag the structure with the mouse to rotate

The history of DFPase is closely linked to the pioneering work of David Nachmannsohn. Nachmannsohn worked on the theory of axonal conduction including the role of the cholinergic synaptic transmission system. Electrophysiology was still limited in the 1950s and 60s, with the modern patch clamp technique not available. To investigate axonal conduction with the available electrodes it was necessary to work with a model species that contained an axon large enough for electrode insertion. Nachmannsohn's group used the calmar Loligo pealei for their experiments and one of these experiments tried to block axonal conduction irreversibly inhibiting the cholinesterases using the potent inhibitor diisopropyl fluorophosphate (DFP). The DFP concentration required to block conduction in the axon turned out to be three orders of magnitude higher than the concentration required to inhibit cholinesterases in solution.

Francis C.G. Hoskin, at that time assistant professor at Columbia University NY, tried to investigate this surprising behavior by using 14C labeled DFP. He was able to show that radioactivity rapidly accumulated in the interior of the axon. But the compound found was not DFP but diisopropyl phosphoric acid. He concluded that the axonal envelope contains a potent enzyme that accounts for the high concentrations of DFP required to block conduction[1]. Hoskin continued his work after becoming full professor at the Illinois Institute of Technology in Chicago. He spent his summer months at the Marine Biological Laboratory at Woodshole on the Cape Cod peninsula preparing enzyme from squid tissue. Apart from the axon, DFPase is also found in the hepatopancreas, saliva and head ganglion[2][3]. He demonstrated that the enzyme does not only hydrolyze DFP, but also the nerve agents Sain, Soman and Tabun[4][5]. Efforts to isolate the complete gene of DFPase from Loligo pealei were unfortunately not successful[6].

DFPase from Loligo vulgaris

Prof. Heinz Rüterjans from the University of Frankfurt in Germany showed great interest in the squid enzmye after conducting some research on an OP hydrolase from hog kidney. While Loligo pealei was readily available in the waters around Woodshole, the Frankfurt lab worked with the Mediterranain squid Loligo vulgaris. Experiments with monoclonal antibodies reveals that the enzymes from both species are very similar although not identical[7]. The group of Prof Rüterjans was to create a cDNA bank of the squid and to finally isolate the DFPase gene. Subsequently the gene was cloned into E. coli and enzyme yields from a liter culture reached more than 100 mg/l after optimal expression conditions were found[8].

In parallel to kinetic studies and studies of the effects of chemical modifications on the protein[9], structural investigations were initiated. The first X-ray structure was solved at a resolution of 1.8 Å (PDB: 1e1a)[10]. Subsequent determinations of the DFPase structure were able to push the resolution to 0.85 Å (PDB: 1pjx)[11]. At this resolution individual atoms are visible in the electron density maps as well as some normally invisible hydrogen atoms.

DFPase from Loligo vulgaris consists of 314 amino acids and contains two calcium ions. The overall structure resembles a sixfold β-propeller with a central water filled tunnel. A high affinity calcium ion is located in the center of the molecule and is important to maintain the structural integrity of the protein. The second low-affinity calcium ion is located at the base of the active site, sealing the water filled tunnel, and is important for catalysis. The role of the catalytic calcium ion was demonstrated by the removal of this ion that resulted in a folded but inactive enzyme[12] and also by site-directed mutagenesis[13]. Inspection of the active site shows the calcium coordinated by four amino acid residues and a total coordination number of seven. The three remaining ligands are water molecules. Two of them are located below the metal ion forming the "dead end" of the central water tunnel and one is located on top of the metal ion in the active site. The DFPase wild-type efficiently hydrolyzes DFP and G-type nerve agents including Sarin, Soman, Cyclosarin and Tabun. However, DFPase does not catalyze the hydrolysis of VX and Paraoxon.

Reaction mechanism of DFPase

The first reaction mechanism of DFPase was proposed at the same time the X-ray structure was published[14]. An obvious candidate for a water-activating residue was histidine 287. In fact, mutant H287N turned out to retain only a small residual activity. So the first proposed mechanism argued that the incoming substrate DFP would replace the calcium coordinating water molecule in the active site and the metal would function as an electrophile making the phosphorus atom of DFP more susceptible for nucleophilic attack by water activated by H287.

This mechanism was challenged when mutations of DFPase (H287F and H287L) were generated that still maintained 65-80% of the wild-type activity[15]. Computational docking of DFP in the DFPase active site also revealed that the orientation of the DFP molecule with the fluoride leading group pointing away from H287 as required for an inline attack of the activated water was energetically unfavorable. Other point mutations did not reveal any other amino acid residue responsible for water activation. A new mechanism was proposed based on new experimental findings[16].

3D Structures of squid DFPase

  • 1e1a This is the original structure solved at 1.8 Å (cryo conditions)
  • 1pjx Atomic resolution structure of DFPase solved at 0.85 Å.
  • 2gvw Structure solved at at 1.86 Å (room temperature).
  • 3byc This is the neutron structure of DFPase solved by joint X-ray and neutron refinement.
  • 3kgg X-ray structure of perdeuterated DFPase at 2.1 Å (room temperature).
  • 2gvv DFPase in complex with dicyclopentylphosphoroamidate (DcPPA)
  • 3li3 DFPase mutant D121E
  • 3li4 DFPase mutant N120D/N175D/D229N
  • 3li5 DFPase mutant E21Q/N120D/N175D/D229N
  • 3hlh DFPase mutant E37A/Y144A/R146A/T195M
  • 3hli DFPase mutant E37D/Y144A/R146A/T195M
  • 2gvu DFPase mutant D229N/N120D
  • 2gvx DFPase mutant D229N/N175D
  • 2iax DFPase mutant D232S
  • 2iaw DFPase mutant D175D
  • 2iav DFPase mutant H287A
  • 2iau DFPase mutant W244Y
  • 2iat DFPase mutant W244L
  • 2ias DFPase mutant W244F
  • 2iar DFPase mutant W244H
  • 2iaq DFPase mutant S271A
  • 2iap DFPase mutant E21Q
  • 2iao DFPase mutant E37Q

References & Notes

  1. ↑ Hoskin FC, Rosenberg P, Brzin M. Re-examination of the effect of DFP on electrical and cholinesterase activity of squid giant axon. Proc Natl Acad Sci U S A. 1966 May;55(5):1231-5. PMID:5225521
  2. ↑ Hoskin FC, Long RJ. Purification of a DFP-hydrolyzing enzyme from squid head ganglion. Arch Biochem Biophys. 1972 Jun;150(2):548-55. PMID:4339736
  3. ↑ Garden JM, Hause SK, Hoskin FC, Roush AH. Comparison of DFP-hydrolyzing enzyme purified from head ganglion and hepatopancreas of squid (Loligo pealei) by means of isoelectric focusing. Comp Biochem Physiol C. 1975 Dec 1;52(2):95-8. PMID:3372
  4. ↑ Hoskin FC. Diisopropylphosphorofluoridate and Tabun: enzymatic hydrolysis and nerve function. Science. 1971 Jun 18;172(989):1243-5. PMID:5576158
  5. ↑ Hoskin FC, Roush AH. Hydrolysis of nerve gas by squid-type diisopropyl phosphorofluoridate hydrolyzing enzyme on agarose resin. Science. 1982 Mar 5;215(4537):1255-7. PMID:7058344
  6. ↑ Kopec-Smyth K, Deschamps JR, Loomis LD, Ward KB. A partial primary structure of squid hepatopancreas organophosphorus acid anhydrolase. Chem Biol Interact. 1993 Jun;87(1-3):49-54. PMID:8393747
  7. ↑ Deschamps JR, Kopec-Smyth K, Poppino JL, Futrovsky SL, Ward KB. Comparison of organophosphorous acid anhydrolases from different species using monoclonal antibodies. Comp Biochem Physiol C. 1993 Nov;106(3):765-8. PMID:7508356
  8. ↑ Hartleib J, Ruterjans H. High-yield expression, purification, and characterization of the recombinant diisopropylfluorophosphatase from Loligo vulgaris. Protein Expr Purif. 2001 Feb;21(1):210-9. PMID:11162408 doi:10.1006/prep.2000.1360
  9. ↑ Hartleib J, Ruterjans H. Insights into the reaction mechanism of the diisopropyl fluorophosphatase from Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis. Biochim Biophys Acta. 2001 Apr 7;1546(2):312-24. PMID:11295437
  10. ↑ Scharff EI, Koepke J, Fritzsch G, Lucke C, Ruterjans H. Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris. Structure. 2001 Jun;9(6):493-502. PMID:11435114
  11. ↑ Koepke J, Scharff EI, Lucke C, Ruterjans H, Fritzsch G. Statistical analysis of crystallographic data obtained from squid ganglion DFPase at 0.85 A resolution. Acta Crystallogr D Biol Crystallogr. 2003 Oct;59(Pt 10):1744-54. Epub 2003, Sep 19. PMID:14501113
  12. ↑ Hartleib J, Geschwindner S, Scharff EI, Ruterjans H. Role of calcium ions in the structure and function of the di-isopropylfluorophosphatase from Loligo vulgaris. Biochem J. 2001 Feb 1;353(Pt 3):579-89. PMID:11171055
  13. ↑ Blum MM, Chen JC. Structural characterization of the catalytic calcium-binding site in diisopropyl fluorophosphatase (DFPase)--comparison with related beta-propeller enzymes. Chem Biol Interact. 2010 Sep 6;187(1-3):373-9. Epub 2010 Mar 3. PMID:20206152 doi:10.1016/j.cbi.2010.02.043
  14. ↑ Scharff EI, Koepke J, Fritzsch G, Lucke C, Ruterjans H. Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris. Structure. 2001 Jun;9(6):493-502. PMID:11435114
  15. ↑ Katsemi V, Lucke C, Koepke J, Lohr F, Maurer S, Fritzsch G, Ruterjans H. Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme. Biochemistry. 2005 Jun 28;44(25):9022-33. PMID:15966726 doi:10.1021/bi0500675
  16. ↑ Blum MM, Lohr F, Richardt A, Ruterjans H, Chen JC. Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism. J Am Chem Soc. 2006 Oct 4;128(39):12750-7. PMID:17002369 doi:10.1021/ja061887n

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