The DFPase from Loligo vulgaris in sugar surfactant-based bicontinuous microemulsions: structure, dynamics, and enzyme activity.

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is of great interest because of its ability to catalyze the hydrolysis of highly toxic organophosphates. In this work, the enzyme structure in solution (native state) was studied by use of different scattering methods. The results are compared with those from hydrodynamic model calculations based on the DFPase crystal structure. Bicontinuous microemulsions made of sugar surfactants are discussed as host systems for the DFPase. The microemulsion remains stable in the presence of the enzyme, which is shown by means of scattering experiments. Moreover, activity assays reveal that the DFPase still has high activity in this complex reaction medium. To complement the scattering experiments cryo-SEM was also employed to study the microemulsion structure.

Temperature dependence of the surfactant film bending elasticity in a bicontinuous sugar surfactant based microemulsion: a quasielastic scattering study.

Currently, the design of microemulsions is focussed on the formulation of environmentally compatible systems formed by non-harmful amphiphiles and oils. The use of sugar-based surfactants allows the design of microemulsions where, instead of the temperature, the addition of short- or medium-chain alcohols tunes the curvature of the amphiphilic interface. In this work, the resulting temperature stability of a sugar surfactant and rapeseed methyl ester based bicontinuous microemulsion is exploited to study the influence of temperature variations on the bending elastic constant κ. Quasi-elastic scattering of light and neutrons is used to separate long-range collective motions and local thermally excited undulations of the interface. κ in units of kT is found to be independent of temperature over a wide range.

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy.

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P-O-R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert-Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).

Photocatalytic oxidation of organic pollutants on titania-clay composites.

TiO2/Ca-montmorillonite composites were prepared by wet grinding in an agate mill. Positively charged TiO2 nanoparticles are bound to the surface of the negatively charged montmorillonite layers via heterocoagulation; the clay mineral is used as adsorbent and support for the photooxidation process. Aquatic solution of 0.5mM phenol was degraded by irradiation with UV-VIS light (lambda=250-440 and 540-590 nm) in suspensions of TiO2-clay composites and significant photodegradation was observed at 40-60% TiO2/Ca-montmorillonite compositions. Synergistic effect was detected at solid/liquid interface for degradation of phenol and at solid/gas interface in the recycling flow reactors for photooxidation of ethanol and toluene vapors.

Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism.

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).