The Experts below are selected from a list of 141 Experts worldwide ranked by ideXlab platform

Marcmichael Blum - One of the best experts on this subject based on the ideXlab platform.

  • An enzyme containing microemulsion based on skin friendly oil and surfactant as decontamination medium for organo phosphates: phase behavior, structure, and enzyme activity.
    Journal of colloid and interface science, 2013
    Co-Authors: Ralf Stehle, Stefan Wellert, Andre Richardt, Kai Kehe, Jurgen Gab, Marcmichael Blum, Christoph Schulreich, Alain Lapp, Thomas Hellweg
    Abstract:

    The present contribution presents a microemulsion system containing cosmetic oil and sugar surfactant and the enzyme Diisopropyl Fluorophosphatase (DFPase) as active agent for the decontamination of human skin. The bicontinuous structure and the physical properties of the microemulsion are characterized by dynamic light scattering and small angle neutron scattering. The DFPase from the squid Loligo vulgaris is catalyzing the hydrolysis of highly toxic organophosphates. The effect of the enzyme on the structure of the microemulsion is investigated. Moreover, the enzyme/microemulsion system is also studied with respect to its activity using nuclear magnetic resonance spectroscopy leading to promising results. A fast decomposition of the nerve agent sarin is achieved.

  • In vitro and in vivo efficacy of PEGylated Diisopropyl Fluorophosphatase (DFPase).
    Drug testing and analysis, 2011
    Co-Authors: Marco Melzer, Kai Kehe, Anne Heidenreich, Jurgen Gab, Frédéric Dorandeu, Horst Thiermann, Thomas Letzel, Marcmichael Blum
    Abstract:

    Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl Fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization – time of flight – mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD50 dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose. Copyright © 2011 John Wiley & Sons, Ltd.

  • The DFPase from Loligo vulgaris in sugar surfactant-based bicontinuous microemulsions: structure, dynamics, and enzyme activity.
    European biophysics journal : EBJ, 2011
    Co-Authors: Stefan Wellert, Olaf Holderer, Andre Richardt, Jurgen Gab, Marcmichael Blum, Christoph Schulreich, Alain Lapp, Brigtte Tiersch, Joachim Koetz, Ralf Stehle
    Abstract:

    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.

  • Neutron structure and mechanistic studies of Diisopropyl Fluorophosphatase (DFPase).
    Acta crystallographica. Section D Biological crystallography, 2010
    Co-Authors: Julian C H Chen, Paul Langan, Marat Mustyakimov, Benno P Schoenborn, Marcmichael Blum
    Abstract:

    Diisopropyl Fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active-site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal-assisted water activation. These data also allowed for the re-engineering of DFPase through rational design to bind and productively orient the more toxic S(P) stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.

  • X-ray structure of perdeuterated Diisopropyl Fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2010
    Co-Authors: Marcmichael Blum, Paul Langan, Heinz Ruterjans, Benno P Schoenborn, Stephen J. Tomanicek, Harald John, B. Leif Hanson, Julian C H Chen
    Abstract:

    The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated Diisopropyl Fluorophosphatase (DFPase) is reported at 2.1 A resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallo­graphic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 A resolution appears to be a strong predictor of successful neutron structures.

Julian C H Chen - One of the best experts on this subject based on the ideXlab platform.

  • Neutron structure and mechanistic studies of Diisopropyl Fluorophosphatase (DFPase).
    Acta crystallographica. Section D Biological crystallography, 2010
    Co-Authors: Julian C H Chen, Paul Langan, Marat Mustyakimov, Benno P Schoenborn, Marcmichael Blum
    Abstract:

    Diisopropyl Fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active-site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal-assisted water activation. These data also allowed for the re-engineering of DFPase through rational design to bind and productively orient the more toxic S(P) stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.

  • X-ray structure of perdeuterated Diisopropyl Fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2010
    Co-Authors: Marcmichael Blum, Paul Langan, Heinz Ruterjans, Benno P Schoenborn, Stephen J. Tomanicek, Harald John, B. Leif Hanson, Julian C H Chen
    Abstract:

    The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated Diisopropyl Fluorophosphatase (DFPase) is reported at 2.1 A resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallo­graphic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 A resolution appears to be a strong predictor of successful neutron structures.

  • Structural characterization of the catalytic calcium-binding site in Diisopropyl Fluorophosphatase (DFPase)--comparison with related beta-propeller enzymes.
    Chemico-biological interactions, 2010
    Co-Authors: Marcmichael Blum, Julian C H Chen
    Abstract:

    The calcium-dependent phosphotriesterase Diisopropyl Fluorophosphatase (DFPase) from the squid Loligo vulgaris efficiently hydrolyzes a wide range of organophosphorus nerve agents. The two calcium ions within DFPase play essential roles for its function. The lower affinity calcium ion located at the bottom of the active site participates in the reaction mechanism, while the high affinity calcium in the center of the protein maintains structural integrity of the enzyme. The activity and structures of three DFPase variants targeting the catalytic calcium-binding site are reported (D121E, N120D/N175D/D229N, and E21Q/N120D/N175D/D229N), and the effect of these mutations on the overall structural dynamics of DFPase is examined using molecular dynamics simulations. While D229 is crucial for enzymatic activity, E21 is essential for calcium binding. Although at least two negatively charged side chains are required for calcium binding, the addition of a third charge significantly lowers the activity. Furthermore, the arrangement of these charges in the binding site is important for enzymatic activity. These results, together with earlier mutational, structural, and kinetic studies, show a highly evolved calcium-binding environment, with a specific electrostatic topology crucial for activity. A number of structural homologues of DFPase have been recently identified, including a chimeric variant of Paraoxonase 1 (PON1), drug resistance protein 35 (Drp35) from Staphylococcus aureus and the gluconolactonase XC5397 from Xanthomonas campestris. Surprisingly, despite low sequence identity, these proteins share remarkably similar calcium-binding environments to DFPase.

  • reversed enantioselectivity of Diisopropyl Fluorophosphatase against organophosphorus nerve agents by rational design
    Journal of the American Chemical Society, 2009
    Co-Authors: Marco Melzer, Kai Kehe, Julian C H Chen, Anne Heidenreich, Jurgen Gab, Marianne Koller, Marcmichael Blum
    Abstract:

    Diisopropyl Fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate Diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.

  • Rapid determination of hydrogen positions and protonation states of Diisopropyl Fluorophosphatase by joint neutron and X-ray diffraction refinement
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Marcmichael Blum, Paul Langan, Kai Kehe, Heinz Ruterjans, Marat Mustyakimov, Benno P Schoenborn, Julian C H Chen
    Abstract:

    Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of Diisopropyl Fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca–O–H angle is 53°, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.

Jurgen Gab - One of the best experts on this subject based on the ideXlab platform.

  • An enzyme containing microemulsion based on skin friendly oil and surfactant as decontamination medium for organo phosphates: phase behavior, structure, and enzyme activity.
    Journal of colloid and interface science, 2013
    Co-Authors: Ralf Stehle, Stefan Wellert, Andre Richardt, Kai Kehe, Jurgen Gab, Marcmichael Blum, Christoph Schulreich, Alain Lapp, Thomas Hellweg
    Abstract:

    The present contribution presents a microemulsion system containing cosmetic oil and sugar surfactant and the enzyme Diisopropyl Fluorophosphatase (DFPase) as active agent for the decontamination of human skin. The bicontinuous structure and the physical properties of the microemulsion are characterized by dynamic light scattering and small angle neutron scattering. The DFPase from the squid Loligo vulgaris is catalyzing the hydrolysis of highly toxic organophosphates. The effect of the enzyme on the structure of the microemulsion is investigated. Moreover, the enzyme/microemulsion system is also studied with respect to its activity using nuclear magnetic resonance spectroscopy leading to promising results. A fast decomposition of the nerve agent sarin is achieved.

  • In vitro and in vivo efficacy of PEGylated Diisopropyl Fluorophosphatase (DFPase).
    Drug testing and analysis, 2011
    Co-Authors: Marco Melzer, Kai Kehe, Anne Heidenreich, Jurgen Gab, Frédéric Dorandeu, Horst Thiermann, Thomas Letzel, Marcmichael Blum
    Abstract:

    Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl Fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization – time of flight – mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD50 dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose. Copyright © 2011 John Wiley & Sons, Ltd.

  • The DFPase from Loligo vulgaris in sugar surfactant-based bicontinuous microemulsions: structure, dynamics, and enzyme activity.
    European biophysics journal : EBJ, 2011
    Co-Authors: Stefan Wellert, Olaf Holderer, Andre Richardt, Jurgen Gab, Marcmichael Blum, Christoph Schulreich, Alain Lapp, Brigtte Tiersch, Joachim Koetz, Ralf Stehle
    Abstract:

    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.

  • Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of Diisopropyl Fluorophosphatase (DFPase) with ^1H–^31P HSQC NMR spectroscopy
    Analytical and Bioanalytical Chemistry, 2010
    Co-Authors: Jurgen Gab, Stefan Wellert, Thomas Hellweg, Kai Kehe, Marco Melzer, Marcmichael Blum
    Abstract:

    The enzyme Diisopropyl Fluorophosphatase (DFPase, EC 3.1.8.2) 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. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D ^1H–^31P HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 μL), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 μmol L^−1 on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment. Figure Staggered view of consecutive 1D ^1H–^31P HSQC NMR spectra recorded during the hydrolysis of GF catalyzed by DFPase in aqueous buffer solution. During the course of the enzyme-catalyzed reaction the doublet of doublets (dd) signal of the nerve agent is decreasing and at the same time the doublet (d) signal of the phosphonate is increasing. The phosphonate (d) signal is used for quantification because of a better signal-to-noise ratio compared with the (dd) of the agent

  • Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of Diisopropyl Fluorophosphatase (DFPase) with 1H-31P HSQC NMR spectroscopy.
    Analytical and bioanalytical chemistry, 2009
    Co-Authors: Jurgen Gab, Stefan Wellert, Thomas Hellweg, Kai Kehe, Marco Melzer, Marcmichael Blum
    Abstract:

    The enzyme Diisopropyl Fluorophosphatase (DFPase, EC 3.1.8.2) 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. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D H-1-P-31 HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 mu L), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 mu mol L-1 on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment.

Kai Kehe - One of the best experts on this subject based on the ideXlab platform.

  • An enzyme containing microemulsion based on skin friendly oil and surfactant as decontamination medium for organo phosphates: phase behavior, structure, and enzyme activity.
    Journal of colloid and interface science, 2013
    Co-Authors: Ralf Stehle, Stefan Wellert, Andre Richardt, Kai Kehe, Jurgen Gab, Marcmichael Blum, Christoph Schulreich, Alain Lapp, Thomas Hellweg
    Abstract:

    The present contribution presents a microemulsion system containing cosmetic oil and sugar surfactant and the enzyme Diisopropyl Fluorophosphatase (DFPase) as active agent for the decontamination of human skin. The bicontinuous structure and the physical properties of the microemulsion are characterized by dynamic light scattering and small angle neutron scattering. The DFPase from the squid Loligo vulgaris is catalyzing the hydrolysis of highly toxic organophosphates. The effect of the enzyme on the structure of the microemulsion is investigated. Moreover, the enzyme/microemulsion system is also studied with respect to its activity using nuclear magnetic resonance spectroscopy leading to promising results. A fast decomposition of the nerve agent sarin is achieved.

  • In vitro and in vivo efficacy of PEGylated Diisopropyl Fluorophosphatase (DFPase).
    Drug testing and analysis, 2011
    Co-Authors: Marco Melzer, Kai Kehe, Anne Heidenreich, Jurgen Gab, Frédéric Dorandeu, Horst Thiermann, Thomas Letzel, Marcmichael Blum
    Abstract:

    Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl Fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization – time of flight – mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD50 dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose. Copyright © 2011 John Wiley & Sons, Ltd.

  • Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of Diisopropyl Fluorophosphatase (DFPase) with ^1H–^31P HSQC NMR spectroscopy
    Analytical and Bioanalytical Chemistry, 2010
    Co-Authors: Jurgen Gab, Stefan Wellert, Thomas Hellweg, Kai Kehe, Marco Melzer, Marcmichael Blum
    Abstract:

    The enzyme Diisopropyl Fluorophosphatase (DFPase, EC 3.1.8.2) 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. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D ^1H–^31P HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 μL), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 μmol L^−1 on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment. Figure Staggered view of consecutive 1D ^1H–^31P HSQC NMR spectra recorded during the hydrolysis of GF catalyzed by DFPase in aqueous buffer solution. During the course of the enzyme-catalyzed reaction the doublet of doublets (dd) signal of the nerve agent is decreasing and at the same time the doublet (d) signal of the phosphonate is increasing. The phosphonate (d) signal is used for quantification because of a better signal-to-noise ratio compared with the (dd) of the agent

  • Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of Diisopropyl Fluorophosphatase (DFPase) with 1H-31P HSQC NMR spectroscopy.
    Analytical and bioanalytical chemistry, 2009
    Co-Authors: Jurgen Gab, Stefan Wellert, Thomas Hellweg, Kai Kehe, Marco Melzer, Marcmichael Blum
    Abstract:

    The enzyme Diisopropyl Fluorophosphatase (DFPase, EC 3.1.8.2) 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. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D H-1-P-31 HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 mu L), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 mu mol L-1 on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment.

  • reversed enantioselectivity of Diisopropyl Fluorophosphatase against organophosphorus nerve agents by rational design
    Journal of the American Chemical Society, 2009
    Co-Authors: Marco Melzer, Kai Kehe, Julian C H Chen, Anne Heidenreich, Jurgen Gab, Marianne Koller, Marcmichael Blum
    Abstract:

    Diisopropyl Fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate Diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.

Paul Langan - One of the best experts on this subject based on the ideXlab platform.

  • Hydrolysis of DFP and the Nerve Agent (S)‑Sarin by DFPase Proceeds along Two Different Reaction Pathways: Implications for Engineering Bioscavengers
    2015
    Co-Authors: Troy Wymore, Marilyn J Field, Paul Langan, Jeremy C. Smith, J. Michael Parks
    Abstract:

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents (“bioscavengers”) is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, Diisopropyl Fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of Diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P–F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the Diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds

  • Hydrolysis of DFP and the nerve agent (S)-sarin by DFPase proceeds along two different reaction pathways: implications for engineering bioscavengers.
    Journal of Physical Chemistry B, 2014
    Co-Authors: Troy Wymore, Marilyn J Field, Paul Langan, Jeremy C. Smith, J. Michael Parks
    Abstract:

    Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ("bioscavengers") is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, Diisopropyl Fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of Diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P-F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca(2+), donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the Diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds.

  • Neutron structure and mechanistic studies of Diisopropyl Fluorophosphatase (DFPase).
    Acta crystallographica. Section D Biological crystallography, 2010
    Co-Authors: Julian C H Chen, Paul Langan, Marat Mustyakimov, Benno P Schoenborn, Marcmichael Blum
    Abstract:

    Diisopropyl Fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active-site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal-assisted water activation. These data also allowed for the re-engineering of DFPase through rational design to bind and productively orient the more toxic S(P) stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.

  • X-ray structure of perdeuterated Diisopropyl Fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2010
    Co-Authors: Marcmichael Blum, Paul Langan, Heinz Ruterjans, Benno P Schoenborn, Stephen J. Tomanicek, Harald John, B. Leif Hanson, Julian C H Chen
    Abstract:

    The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated Diisopropyl Fluorophosphatase (DFPase) is reported at 2.1 A resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallo­graphic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 A resolution appears to be a strong predictor of successful neutron structures.

  • Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules
    Acta Crystallographica Section D Biological Crystallography, 2009
    Co-Authors: Paul D. Adams, Marat Mustyakimov, Pavel V. Afonine, Paul Langan
    Abstract:

    X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallo­graphic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for Diisopropyl Fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains.