User:Marc-Michael Blum/Sandbox1

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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

WThe 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].

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

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Marc-Michael Blum

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