The organophosphate acid anhydrolase (OPAA) is a member of a class of bimetalloenzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus compounds, including fluorine-containing chemical nerve agents. It also belongs to a family of prolidases, with significant activity against various Xaa-Pro dipeptides. Here we report the X-ray structure determination of the native OPAA (58 kDa mass) from Alteromonas sp. strain JD6.5 and its cocrystal with the inhibitor mipafox [N,N'-diisopropyldiamidofluorophosphate (DDFP)], a close analogue of the nerve agent organophosphate substrate diisopropyl fluorophosphate (DFP). The OPAA structure is composed of two domains, amino and carboxy domains, with the latter exhibiting a "pita bread" architecture and harboring the active site with the binuclear Mn(2+) ions. The native OPAA structure revealed unexpectedly the presence of a well-defined nonproteinaceous density in the active site whose identity could not be definitively established but is suggestive of a bound glycolate, which is isosteric with a glycine (Xaa) product. All three glycolate oxygens coordinate the two Mn(2+) atoms. DDFP or more likely its hydrolysis product, N,N'-diisopropyldiamidophosphate (DDP), is present in the cocrystal structure and bound by coordinating the binuclear metals and forming hydrogen bonds and nonpolar interactions with active site residues. An unusual common feature of the binding of the two ligands is the involvement of only one oxygen atom of the glycolate carboxylate and the product DDP tetrahedral phosphate in bridging the two Mn(2+) ions. Both structures provide new understanding of ligand recognition and the prolidase and organophosphorus hydrolase catalytic activities of OPAA.
Efficacy of chlorine dioxide (CD) gas generated by two distinct generation systems, Sabre (wet system with gas generated in water) and ClorDiSys (dry system with gas generated in air), was evaluated for inactivation of Bacillus anthracis spores on six building interior surfaces. The six building materials included carpet, acoustic ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. There was no statistically significant difference in the data due to the CD generation technology at a 95% confidence level. Note that a common method of CD gas measurement was used for both wet and dry CD generation types. Doses generated by combinations of different concentrations of CD gas (500, 1,000, 1,500, or 3,000 parts per million of volume [ppmv]) and exposure times (ranging between 0.5 and 12 h) were used to evaluate the relative role of fumigant exposure period and total dose in the decontamination of building surfaces. The results showed that the time required to achieve at least a 6-log reduction in viable spores is clearly a function of the material type on which the spores are inoculated. The wood and cinder block coupons required a longer exposure time to achieve a 6-log reduction. The only material showing a clear statistical difference in rate of decay of viable spores as a function of concentration was cinder block. For all other materials, the profile of spore kill (i.e., change in number of viable spores with exposure time) was not dependent upon fumigant concentration (500 to 3,000 ppmv). The CD dose required for complete spore kill on biological indicators (typically, 1E6 spores of Bacillus atrophaeus on stainless steel) was significantly less than that required for decontamination of most of the building materials tested.
Public Reporting Burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Approved for Public Release; Government Rights Detection of Organophosphorus Compounds by Covalently Immobilized Organophosphorus HydrolaseReport Title ABSTRACTAs a consequence of organophosphorus (OP) toxins posing a threat to human life globally, organophosphorus hydrolase (OPH) has become the enzyme of choice to detoxify such compounds. Organophosphorus hydrolase was covalently immobilized onto a quartz substrate for utilization in paraoxon detection. The substrate was cleaned and modified prior to chemical attachment. Each modification step was monitored by imaging ellipsometry as the thickness increased with each modification step. The chemically attached OPH was labeled with a fluorescent dye (7-isothiocyanato-4-methylcoumarin) for the detection of paraoxon in aqueous solution, ranging from 1 nanomole to 10 micromole. UV-visible spectra were also acquired for the determination of the hydrolysis product of para-oxon, namely p-nitrophenol.
The secondary structure of the organophosphorus acid anhydrolase (OPAA) Langmuir monolayer in the absence and presence of diisopropylfluorophosphate (DFP) in the subphase was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS (PM-IRRAS). The results of both the IRRAS and the PM-IRRAS indicated that the alpha-helix and the beta-sheet conformations in OPAA were parallel to the air-water interface at a surface pressure of 0 mN.m-1 in the absence of DFP in the subphase. When the surface pressure increased, the alpha-helix and the beta-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 x 10(-5) M, the alpha-helix conformation of OPAA was still parallel to the air-water interface, whereas the beta-sheet conformation was perpendicular at 0 mN.m-1. The orientations of both the alpha-helix and the beta-sheet conformations did not change with the increase of surface pressure. The shape of OPAA molecules is supposed to be elliptic, and the long axis of OPAA was parallel to the air-water interface in the absence of DFP in the subphase, whereas the long axis became perpendicular in the presence of DFP. This result explains the decrease of the limiting molecular area of the OPAA Langmuir monolayer when DFP was dissolved in the subphase.
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