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Title: Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris

Abstract

Diisopropyl fluorophosphatase (DFPase) effectively hydrolyzes a number of organophosphorus nerve agents, including sarin, cyclohexylsarin, soman and tabun. Neutron diffraction data have been collected from DFPase crystals to 2.2 Å resolution in an effort to gain further insight into the mechanism of this enzyme. The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D{sub 2}O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 Å resolution on a relatively small (0.43 mm{sup 3}) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

Authors:
 [1];  [2];  [1];  [3];  [1];  [4];  [4];  [3];  [1]
  1. Institute of Biophysical Chemistry, J. W. Goethe University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt (Germany)
  2. (Germany)
  3. (United States)
  4. Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545 (United States)
Publication Date:
OSTI Identifier:
22360253
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 1; Other Information: PMCID: PMC2330113; PMID: 17183172; PUBLISHER-ID: ll5096; OAI: oai:pubmedcentral.nih.gov:2330113; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ATOMS; CRYSTALS; DIFFUSION; NEUTRON DIFFRACTION; NEUTRONS; REACTION KINETICS; RESOLUTION; SPALLATION; TIME-OF-FLIGHT METHOD

Citation Formats

Blum, Marc-Michael, Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, D-80937 Munich, Koglin, Alexander, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, Rüterjans, Heinz, Schoenborn, Benno, Langan, Paul, Department of Chemistry, University of Toledo, Toledo, OH 53606, and Chen, Julian C.-H., E-mail: chen@chemie.uni-frankfurt.de. Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309106052924.
Blum, Marc-Michael, Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, D-80937 Munich, Koglin, Alexander, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, Rüterjans, Heinz, Schoenborn, Benno, Langan, Paul, Department of Chemistry, University of Toledo, Toledo, OH 53606, & Chen, Julian C.-H., E-mail: chen@chemie.uni-frankfurt.de. Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. United Kingdom. doi:10.1107/S1744309106052924.
Blum, Marc-Michael, Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, D-80937 Munich, Koglin, Alexander, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, Rüterjans, Heinz, Schoenborn, Benno, Langan, Paul, Department of Chemistry, University of Toledo, Toledo, OH 53606, and Chen, Julian C.-H., E-mail: chen@chemie.uni-frankfurt.de. Mon . "Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris". United Kingdom. doi:10.1107/S1744309106052924.
@article{osti_22360253,
title = {Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris},
author = {Blum, Marc-Michael and Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, D-80937 Munich and Koglin, Alexander and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 and Rüterjans, Heinz and Schoenborn, Benno and Langan, Paul and Department of Chemistry, University of Toledo, Toledo, OH 53606 and Chen, Julian C.-H., E-mail: chen@chemie.uni-frankfurt.de},
abstractNote = {Diisopropyl fluorophosphatase (DFPase) effectively hydrolyzes a number of organophosphorus nerve agents, including sarin, cyclohexylsarin, soman and tabun. Neutron diffraction data have been collected from DFPase crystals to 2.2 Å resolution in an effort to gain further insight into the mechanism of this enzyme. The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D{sub 2}O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 Å resolution on a relatively small (0.43 mm{sup 3}) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.},
doi = {10.1107/S1744309106052924},
journal = {Acta Crystallographica. Section F},
number = Pt 1,
volume = 63,
place = {United Kingdom},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Crystals of the blue copper protein amicyanin suitable for neutron diffraction were grown by the sitting-drop method, followed by repeated macroseeding using solutions prepared with D{sub 2}O. Although the crystal sizes were the same, crystals grown using solutions made up in H{sub 2}O in the initial stages of macroseeding and solutions with D{sub 2}O in later stages did not diffract neutrons well. However, when the protein was initially exchanged with buffered D{sub 2}O and then crystallized and also macroseeded using solutions made up in D{sub 2}O throughout, the crystals diffracted neutrons to high resolution. One of those crystals was usedmore » to collect a data set to a resolution of 1.9 {angstrom}.« less
  • In order to investigate the role of the protonation states of protein residues in O{sub 2} binding, large crystals of deoxy HbA (∼20 mm{sup 3}) were grown in D{sub 2}O under anaerobic conditions for neutron diffraction studies. Human hemoglobin (HbA) is an intricate system that has evolved to efficiently transport oxygen molecules (O{sub 2}) from lung to tissue. Its quaternary structure can fluctuate between two conformations, T (tense or deoxy) and R (relaxed or oxy), which have low and high affinity for O{sub 2}, respectively. The binding of O{sub 2} to the heme sites of HbA is regulated by protonsmore » and by inorganic anions. In order to investigate the role of the protonation states of protein residues in O{sub 2} binding, large crystals of deoxy HbA (∼20 mm{sup 3}) were grown in D{sub 2}O under anaerobic conditions for neutron diffraction studies. A time-of-flight neutron data set was collected to 1.8 Å resolution on the Protein Crystallography Station (PCS) at the spallation source run by Los Alamos Neutron Science Center (LANSCE). The HbA tetramer (64.6 kDa; 574 residues excluding the four heme groups) occupies the largest asymmetric unit (space group P2{sub 1}) from which a high-resolution neutron data set has been collected to date.« less
  • The structure and mechanism of diisopropyl fluorophosphatase (DFPase) have been studied using a variety of methods, including isotopic labelling, X-ray crystallography and neutron crystallography. The neutron structure of DFPase, mechanistic studies and subsequent rational design efforts are described. 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 ascertainmore » 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{sub P} stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.« less
  • Time-of-flight neutron powder diffraction data have been obtained in situ at temperatures up to 1,273 K for two samples of MgAl{sub 2}O{sub 4} (spinel). The samples were synthesized under different experimental conditions (single-crystal boule vs. air-quenched sintered pellet) and exhibited different degrees of cation disorder at room temperature. The Rietveld method was used to fit the data which were obtained initially at room temperature and then at various temperatures as the samples were cooled from 1,273 K. Both samples exhibit statistically equivalent degrees of disorder in the temperature range 973-1,273 K. The atomic fraction of Al in the tetrahedral sitemore » increases from 0.25(1) at 973 K to 0.35(2) at 1,273 K. This amount of disorder is significantly less than that determined by an earlier nuclear magnetic resonance experiment. The slight nonuniform variation of the atomic parameters in this temperature range for both samples may suggest a behavior that is inconsistent with a simple process of cation reordering on cooling.« less