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

Abstract

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 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 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.

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

“…The crystal used here was 0.43 mm 3 in size, among the smallest sample size used so far for neutron crystallography (36). The crystal was exchanged for less than 1 month before the start of data acquisition.…”

“…Room-temperature neutron data were collected and processed as reported (36). Briefly, a single crystal measuring 2.4 ϫ 0.5 ϫ 0.36 mm in size was mounted in a glass capillary, and labile hydrogens were exchanged for deuteriums by vapor diffusion for 1 week before the start of data collection.…”

“…The structure was refined by using a version of CNS (12), called nCNS, modified for joint X-ray and neutron refinement (13). The refinement used neutron data between 20.0 -2.2 Å resolution, accounting for a slight difference in the number of reflections than reported (36). A detailed treatment of the joint refinement method is described in the SI Text, and statistics of the joint refinement of DFPase are reported in Table S1.…”

Hydrolytic Enzymes for Chemical Warfare Agent Decontamination

“…A dataset of 2.2 Å resolution was recorded and the structure will reveal more details about the protonation states of amino acid side chains and water molecules in the active site and the central tunnel [26] .…”

Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase

“…The sub-Ångstrom resolution X-ray (0.85Å) structure was previously determined [11]. Congruently, Blum et al , (2009) have performed a neutron diffraction study at 2.2 Å resolution, benefiting thereby from the high diffraction power of the DFPase crystals [16] and obtained a combined neutron-X-ray (X-N) model. Since both sub-Ångstrom X-ray resolution data and medium resolution neutron data are available for DFPase, the respective structures enables cross-comparison between hydrogen bonds and water networks.…”