X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction

Abstract

PDB Reference: perdeuterated diisopropyl fluorophosphatase, 3kgg.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 roomtemperature 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.

Production and characterization of recombinant perdeuterated cholesterol oxidase

“…It is important to ensure that perdeuteration does not affect the overall structure of the protein. Although X-ray structures of perdeuterated proteins generally show no significant differences in the structures compared with nondeuterated proteins [14,26,38,39,46,47], Chatake and coworkers [48] showed there were differences in dissimilatory sulfite reductase D crystals soaked with various deuterium concentrations, although these did not translate to major structural differences. Liu and coworkers [25] did find that a catalytic residue was displaced in the deuterated structure, and as a consequence a water molecule, which is suspected to play a role in the catalysis of haloalkane dehalogenase, was not present in the perdeuterated structure, indicating that it is important to determine that perdeuteration does not affect the protein structure.…”

Structural Basis of the γ-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

“…In the course of the crystallographic refinement, a strong electron density was found inside the β-propeller structure in both mouse and human SMP30/GNL. Earlier structure analysis of relevant molecules [11], [13]–[15] suggested that the strong density represents a divalent metal ion. The ICP-MS analysis suggested that the divalent metal ion bound to SMP30/GNL molecules in the crystal showed heterogeneity; the SMP30/GNL molecules bound Mn 2+ , Zn 2+ , Mg 2+ , or Ca 2+ in the crystal ( Table S3 ).…”

“…The crystal structure of mouse SMP30/GNL was compared with other related enzymes, human SMP30/GNL [11], DFPase [13], Drp35 [14], PON [15], and a putative GNL (PDB ID: 3E5Z) in PDB. The overall folds of these proteins were essentially the same, i.e., they adopted a six-bladed β-propeller structure.…”

“…The crystal structure showed that human SMP30/GNL adopts a six-bladed β-propeller fold [12], which is similar to those of squid diisopropyl-fluorophosphatase (DFPase) [13], drug resistance protein 35 (Drp35) [14], and paraoxonase (PON) [15]. These proteins show an amino acid sequence similarity with human SMP30/GNL.…”

“…While the formation of the γ-lactone ring is a critical step in the synthesis of ascorbic acid, the catalytic mechanism of the γ-lactone-ring formation remains elusive due to the lack of structural information of the substrate complex of SMP30/GNL. Furthermore, while some of the related enzyme structures have been determined [13]–[15], no structural information has been obtained for substrate complexes relevant to the GNL activity. Here, we report the crystal structures of mouse SMP30/GNL not only in the substrate-free form but also in the substrate/product-analogue complex forms ( Figure 1B, C, D ).…”