Plot of C-α RMSD values between d-DFPase and h-DFPase

Protein structures solved by neutron diffraction have the significant advantage compared to X-ray structures that hydrogen atoms are clearly observable in the nuclear density maps. This allows the determination of protonation states in amino acid side-chains and the orientation of solvent molecules (especially water) in space. This is of special importance for the elucidation of enzyme mechanisms. The disadvantages of neutron diffraction with proteins exist in the small number of powerful neutron sources and dedicated instruments worldwide. Also – neutron flux even at the most powerful sources is small compared to photon flux at X-ray sources. Therefore large protein crystals are required and data collection times can easily be in the region of several weeks. Another problem is the signal to noise ratio. Hydrogen atoms in the crystal are major contributors to this problems because the large incoherent scattering cross section of hydrogen. High values for this incoherent scattering cross section lead to diffuse scattering and negatively influence the signal to noise ratio. Deuterium on the other hand displays a significantly smaller value (2.05 b for deuterium compared with 80.27 b for normal hydrogen; 1 b = 100 fm²). To overcome this problem, crystals grown with hydrogenous protein are normally soaked with deuterated mother liquor (or brought in contact via gas diffusion).

Cover of Acta F with DFPase

Labile hydrogens (e.g. those of water in the solvent of in acidic or basic functional groups in the protein) are exchanged with deuterium. Non-labile hydrogens like those in aliphatic or aromatic C-H bonds are no exchanged. Therefore a significant number of hydrogen atoms remain in the protein. Such a partially exchanged crystal was used for the already published neutron structure of DFPase.

To achieve full deuteration of the protein it has to be grown in fully deuterated media. We now report the X-ray structure of fully deuterated DFPase in a new publication in the journal Acta Cryst. F. The structure (solved at room temperature at a resolution of 2.1 Å) shows that full deuteration leads to practically no changes in the protein structure. But even though a very large crystal of d-DFPase was grown (> 2mm³) it did not diffract neutrons beyond very low resolution. An explanation for this unexpected result can be found either in the differences in data aquisition (cross section of the neutron beam compared to the X-ray beam used) or can be based on crystallographic parameters. The scattering characteristics of successful neutron experiments and associated X-ray data are presented in tabulated form and can serve as a guidance for future neutron experiments.

X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): Perdeuteration of proteins for neutron diffraction.
Blum MM, Tomanicek S, John H, Hanson L, Rüterjans H, Schoenborn BP, Langan P, Chen JC.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66(4):379-385.
http://dx.doi.org/10.1107/S1744309110004318

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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 Å 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 Å resolution appears to be a strong predictor of successful neutron structures.

Catalytic calcium binding-site of DFPase

The enzyme DFpase is structurally well characterized. An atomic resolution X-ray structure and a neutron structure are available. In addition to this a number of DFPase mutants were generated and for some of them X-ray structures were determined. What was missing up to now was a systematic investigation of the calcium binding-site of DFPase with respect to catalytic activity and the ability for metal binding. A summary of existing data and three new X-ray structures of mutants changing residues in the calcium binding-site were now published in a paper in the journal Chemico-Biological Interactions. We also deduce rules for activity and metal biding.

A comparison with the structurally related proteins Paraoxonase (PON1), Drug Resistance Protein 35 (Drp35) from S. aureus or the Gluconolactonase XC5397 from Xanthomonas campestris reveal calcium binding sites with highly similar topology but in part different enzymatic activities (PON1 is also a phosphotriesterase but the native substrates of PON1, Drp35 und XC5397 seem to be lactones). The possible applicability of the rules deduced for DFPase to the other proteins is discussed.

Structural characterization of the catalytic calcium binding site in diisopropyl fluorophosphatase (DFPase) and comparison with related β-propeller enzymes.
Blum MM, Chen JC.
Chem. Biol. Interact. 2010; 187(1-3):373-379.
http://dx.doi.org/10.1016/j.cbi.2010.02.043

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.

Mechanism of adduct formation

Buffer compounds are used to maintain a certain pH in solution. Besides buffer capacity and pH stability over prolonged times the non-reactivity with other components of the solution is an important factor for buffer selection. In a new publication in the Journal of Chrmatography B we report the formation of stable adducts of nerve agents like Sarin, Soman or Cyclosain with common and widely employed buffer compounds like TRIS, TES or HEPES. The reaction proceeds in competition with spontaneous hydrolysis in aqueous solution and yields can reach up to 40% based on the organophosphate/phosphonate present. Highest yields are achieved with high buffer concentrations and high pH.

Using a recently presented NMR method to monitor the degradation of nerve agents by enzymes in buffered solution we were able to observe the formation of new, phosphorus containing and stable compounds. Using LC-ESI-MS/MS we were able to show that the compounds are adducts of the buffer and the nerve agent. Buffer compounds like TES or TRIS can act both as nitrogen nucleophiles (via the amino group) and as oxygen nucleophiles (via the oxygen atoms of the hydroxyl groups). The identification of the adducts as phosphodiesters (“O-adducts”) was finally achieved by NMR spectroscopy.

As a potential reaction mechanism we propose that the amino group of the buffer acts as an intramolecular proton acceptor, which can accept a proton from a hydroxyl group of the buffer. This increases the nucleophilicity of the hydroxyl oxygen atom attacking the phosphorus atom of the warfare agent leading to the formation of a phosphodiester (with organophosphonates like Sarin, Soman and Cyclosarin). As alternative buffer compounds for work with nerve agents we propose MOPS (pK = 7.2), CHES (pK = 9.3) und MES (pK = 6.15). These compounds do not contain a combination of hydroxyl and amino groups and do not show any adducts formation in solution.

Stable adducts of nerve agents sarin, soman and cyclosarin with TRIS, TES and related buffer compounds–Characterization by LC-ESI-MS/MS and NMR and implications for analytical chemistry.
Gäb J, John H, Melzer M, Blum MM.
J. Chromatogr. B 2010; 878(17-18):1382-1390.
http://dx.doi.org/10.1016/j.jchromb.2010.01.043

Abstract:
Buffering compounds like TRIS are frequently used in chemical, biochemical and biomedical applications to control pH in solution. One of the prerequisites of a buffer compound, in addition to sufficient buffering capacity and pH stability over time, is its non-reactivity with other constituents of the solution. This is especially important in the field of analytical chemistry where analytes are to be determined quantitatively. Investigating the enzymatic hydrolysis of G-type nerve agents sarin, soman and cyclosarin in buffered solution we have identified stable buffer adducts of TRIS, TES and other buffer compounds with the nerve agents. We identified the molecular structure of these adducts as phosphonic diesters using 1D 1H-31P HSQC NMR and LC-ESI-MS/MS techniques. Reaction rates with TRIS and TES are fast enough to compete with spontaneous hydrolysis in aqueous solution and to yield substantial amounts (up to 20-40%) of buffer adduct over the course of several hours. A reaction mechanism is proposed in which the amino function of the buffer serves as an intramolecular proton acceptor rendering the buffer hydroxyl groups nucleophilic enough for attack on the phosphorus atom of the agents. Results show that similar buffer adducts are formed with a range of hydroxyl and amino function containing buffers including TES, BES, TRIS, BIS-TRIS, BIS-TRIS propane, Tricine, Bicine, HEPES and triethanol amine. It is recommended to use alternative buffers like MOPS, MES and CHES when working with G-type nerve agents especially at higher concentrations and over prolonged times.

A new publication in Analytical and Bioanalytical Chemistry describes the use of 1H-31P HSQC NMR spectroscopy to monitor of the degradation of highly toxic organophosphorus compounds by the enzyme DFPase. The method can be used for methylphosphonates, a group of compounds including nerve agents sarin (GB), soman (GD), cyclosarin (GF) and also VX. The limit of quantitation (LOQ) of the method is around 100 μM when using a 400 MHz NMR spectrometer. The work is founded on previous results from Koskela et al. (Koskela et al., Anal. Chem. 2007; 79:9098-9106) who were able to use the method to detect agents and hydrolysis products in comlex decontamination fluids. We were now able to show that the procedure works not only in the static case but also for dynamic reaction monitoring.

The method is of special relevance for reaction monitoring in complex media. We were able to show that monitoring is also possible in multi-phase systems by using a biodiesel based bicontinuous microemulsion as a model system. Other methods like pH-stat titration or the use of fluoride sensitive electrodes regularly fail in these complex fluids.

Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of disopropyl fluorophosphatase (DFPase) with 1H-31P HSQC NMR spectroscopy.
Gäb J, Melzer M, Kehe K, Wellert S, Hellweg T, Blum MM.
Anal. Bioanal. Chem. 2009;396(3):1213-1221.
http://dx.doi.org/10.1007/s00216-009-3299-2

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 μM 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.

The enzyme Diisopropyl fluorophosphatase (DFPase) catalyses the hydrolysis of the toxic organophosphorus compound Diisopropyl fluorophosphat (DFP) and a range of highly toxic nerve agents of the so called G-series. This class of nerve agents includes compounds like tabun (GA), sarin, (GB), soamn (GD) and cyclosarin (GF). DFP does not contain any stereocenters and is achiral but all mentioned G-type nerve agents have four different substitutents at the central phosphorus atom. Therefore this phosphorus atom is asymmetric and forms a stereocenter. GA, GB, GF are chiral and exist as pair of enantiomers, which relate to each other like left hand and right hand (GD is a more complicated case as there is an additional stereocenter in one of the side chains).

There is no difference between the enantiomers in chemical reactions that take place in an achiral environment. An example for this is the aqueous hydrolysis of the agents at high pH. But when interacting with other chiral molecules like proteins they diplay distinct differences. The main “target” for the nerve agents is the enzyme acteylcholinesterase (AChE), which is inhibited by the nerve agents by the formation of a stable covalent adduct. One of the two enantiomers shows a significantly higher inhibitory power compared to the other, which is only mildly inhibiting or even non-inhibiting. A similar effect is seen when hydrolyzing (and detoxifying) the agents using DFPase as an enzymatic catalyst. Wildtype DFPase prefers the less toxic enantiomer. This is especially problematic for medical application (in vivo or topical) as rapid reduction of toxicity is of special importance. A potential strategy to reverse enantioselectivity of DFPase is to alter the active site of the enzyme by mutagenesis.

Model of the phosphoenzyme intermediate
of DFPase for the substrate sarin

Due to the legal restrictions for handling highly toxic compounds* the standard approach for mutagenesis using evolutionary methods and high-throuput screening of mutant libraries could not be used. Instead we turned to rational protein design. One should note that reversing enantioselectivity of an enzyme (and keeping activity) is not a trivial task and the number of publications dealing with this issues is still limited. Detailed knowledge about the DFPase structure and the reaction mechanism, which is though to proceed via a phosphoenzyme intermediate, formed an invaluable base for planning the mutations.

The constructed mutants finally displayed the desired reversed enantioselectivity without loosing enzymatic activity. The mutants rather showed enhanced activities (up to 8-fold higher in case of GB). For an optimal combination of selectivity , activity and substrate affinity the mutants should be mixed with wildtype DFPase. Using such mixtures total hydrolysis of the agents can be achieved 4-fold faster even at low substrate concentrations (where substrate affinity is an issue) with an even faster reduction in toxicity as in the first phase of the reaction the more toxic enantiomer is hydrolyzed faster.

Structures of the mutants were deposited in the PDB under accession codes 3HLI and 3HLH.

* Work with nerve agents was conducted in accordance with the Chemical Weapons Convention (CWC) at the Bundeswehr Institute for Pharmacology and Toxicology,

Reversed Enantioselectivity of Diisopropyl Fluorophosphatase against Organophosphorus Nerve Agents by Rational Design.
Melzer M, Chen JC, Heidenreich A, Gäb J, Koller M, Kehe K, Blum MM.
J. Am. Chem. Soc. 2009;131(47):17226-17232.
http://dx.doi.org/10.1021/ja905444g

Research Highlight in Nature Chemistry:
http://dx.doi.org/110.1038/nchem.487

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.

Im the past year the number of entries in the Protein Data Base (PDB) has exceeded the number of 50.000. Most of these structure have been determined by X-ray crystallography, a smaller part by Nuclear Magnetic Resonance (NMR). Compared with this, the number of published neutron diffraction structures of proteins is tiny. Even though the first neutron structure of a protein was already solved in 1969 by Benno Schoenborn (sperm whale myoglobin, the same protein that John Kendrew used to determine the first X-ray structure of a protein ten years earlier) only about 20 proteins were characterized by neutron diffraction up to the present day.

Compared to X-ray diffraction the use of neutrons has a clear advantage. While hydrogen atoms are normally invisible in X-ray structures they become visible with neutrons. Knowing the location of hydrogen atoms means that protonation states of amino acid sidechains are known and that the orientation of water molecules can be determined. This is especially important for the understanding of enzyme machanisms. But why are there so few neutron structures? The reason is the very low flux of even modern neutron sources compared to the photon flux in modern synchrotrons used for X-ray crystallography. Therefore large crystals are required and long measurement time are necessary. Also the number of experimental stations dedicated for bio-macromolecules is limited. Currently only three sources allow the work on proteins (one in the USA, one in France and one in Japan).

The determination of the neutron diffraction structure of the enzyme diisopropyl fluorophosphatase (DFPase) was carried out at the spallation neutron source at Los Alamos National Laboratory (LANL) with one of the smallest crystals ever used for protein neutron crystallography. The volume of the crystal was 0.43 mm³. To record a complete data set about one month of beam time was required. The pulsed spallation source in Los Alamos also allowed the use of time-of-flight (TOF) techniques. The structure was deposited in the PDB under accession code 3BYC.

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement.
Blum MM, Mustyakimov M, Rüterjans H, Kehe K, Schoenborn BP, Langan P, Chen JC.
Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):713-8.
http://dx.doi.org/10.1073/pnas.0807842106

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.