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

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).

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement

“…A second calcium ion, Ca1, located at the base of the active site pocket, is crucial for catalysis; its removal results in a folded, yet inactive enzyme (18). The active-site pocket is elongated and primarily hydrophobic in character, with an apparently rigid structure, as only minor conformational rearrangements are observed upon binding of a substrate analogue (19).…”

X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction

“…The expression and purification of hydrogenous DFPase (h-DFPase) as well as the RT X-ray crystal structure (PDB code 2gvw) have been reported previously (Blum et al, 2006). d-DFPase was expressed according to the same protocol, exchanging TB culture medium for a special perdeuterated culture medium developed at Los Alamos National Laboratory called Altone.…”

“…1) is a potent and well characterized Ca 2+ -dependent phosphotriesterase that is capable of hydrolyzing a variety of organophosphorus compounds that act as irreversible inhibitors of acetylcholinesterase (AChE). Substrates include diisopropyl fluorophosphate (DFP) and a range of highly toxic G-type organophosphorus (OP) nerve agents such as sarin (GB), soman (GD) and cyclosarin (GF) that pose a threat to military personnel and civilian populations (Blum et al, 2006(Blum et al, , 2008Scharff et al, 2001). To elucidate the mechanism of DFPase, the 2.2 Å neutron structure of DFPase has been determined (PDB code 3byc) using a deuterated crystal of 0.43 mm 3 in volume (Blum et al, 2009).…”

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) fromLoligo vulgaris

“…A 1.86 Å roomtemperature X-ray data set of DFPase (structure factors and PDB code 2gvw; Blum et al, 2006) is being used together with the recently collected neutron data. Visual inspection of the initial maps show clear signature features for neutron maps.…”

“…However, mutants of the candidate residue His287 have shown little to no loss of activity and eliminated the histidine as the general base in the reaction (Katsemi et al, 2005). More recently, the catalytic calcium-coordinating residue Asp229 was identified as the nucleophile on the basis of structural, kinetic and isotope-labelling experiments and an alternative mechanism involving a phosphoenzyme intermediate was proposed (Blum et al, 2006). A water molecule then attacks the carboxyl C atom of Asp229 to generate the product.…”