2GVX

Structure of diisopropyl fluorophosphatase (DFPase), mutant D229N / N175D


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.257 
  • R-Value Work: 0.216 
  • R-Value Observed: 0.216 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Binding of a Designed Substrate Analogue to Diisopropyl Fluorophosphatase: Implications for the Phosphotriesterase Mechanism

Blum, M.-M.Lohr, F.Richardt, A.Ruterjans, H.Chen, J.C.-H.

(2006) J Am Chem Soc 128: 12750-12757

  • DOI: https://doi.org/10.1021/ja061887n
  • Primary Citation of Related Structures:  
    2GVU, 2GVV, 2GVW, 2GVX

  • PubMed Abstract: 

    A wide range of organophosphorus nerve agents, including Soman, Sarin, and Tabun is efficiently hydrolyzed by the phosphotriesterase enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. To date, the lack of available inhibitors of DFPase has limited studies on its mechanism. The de novo design, synthesis, and characterization of substrate analogues acting as competitive inhibitors of DFPase are reported. The 1.73 A crystal structure of O,O-dicyclopentylphosphoroamidate (DcPPA) bound to DFPase shows a direct coordination of the phosphoryl oxygen by the catalytic calcium ion. The binding mode of this substrate analogue suggests a crucial role for electrostatics in the orientation of the ligand in the active site. This interpretation is further supported by the crystal structures of double mutants D229N/N120D and D229N/N175D, designed to reorient the electrostatic environment around the catalytic calcium. The structures show no differences in their calcium coordinating environment, although they are enzymatically inactive. Additional double mutants E21Q/N120D and E21Q/N175D are also inactive. On the basis of these crystal structures and kinetic and mutagenesis data as well as isotope labeling we propose a new mechanism for DFPase activity. Calcium coordinating residue D229, in concert with direct substrate activation by the metal ion, renders the phosphorus atom of the substrate susceptible for attack of water, through generation of a phosphoenzyme intermediate. Our proposed mechanism may be applicable to the structurally related enzyme paraoxonase (PON), a component of high-density lipoprotein (HDL).


  • Organizational Affiliation

    Institute of Biophysical Chemistry, J.W. Goethe University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt, Germany.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
diisopropyl fluorophosphatase314Loligo vulgarisMutation(s): 0 
EC: 3.1.8.2
UniProt
Find proteins for Q7SIG4 (Loligo vulgaris)
Explore Q7SIG4 
Go to UniProtKB:  Q7SIG4
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ7SIG4
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.257 
  • R-Value Work: 0.216 
  • R-Value Observed: 0.216 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 42.6α = 90
b = 82.7β = 90
c = 86.7γ = 90
Software Package:
Software NamePurpose
CNSrefinement
PDB_EXTRACTdata extraction
ADSCdata collection
MOSFLMdata reduction
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2006-09-19
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2024-02-14
    Changes: Data collection, Database references, Derived calculations