Browsing by Subject "organophosphates"
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Item Directed evolution of phosphotriesterase for detoxification of the nerve agent VX(Texas A&M University, 2006-10-30) Ghanem, Eman MohamedPhosphotriesterase (PTE) isolated from the soil bacterium Flavobacterium sp. is a member of the amidohydrolase superfamily. PTE catalyzes the hydrolysis of a broad spectrum of organophosphate triesters including the insecticide paraoxon, and the chemical warfare agents; GF, sarin, and soman. In addition, PTE has been shown to catalytically hydrolyze the lethal nerve agent, VX. However, the rate of VX hydrolysis is significantly slower. PTE was subjected to directed evolution studies to identify variants with enhanced activity towards VX hydrolysis. First generation libraries targeted amino acid residues in the substrate binding site. The H254A mutation displayed a 4-fold enhancement in kcat and a 2-fold enhancement in kcat/Km over wild type PTE. The double mutant H254Q/H257F was isolated from the second generation libraries and displayed a 10-fold enhancement in kcat and a 3-fold enhancement in kcat/Km. In addition, H254Q/H257F displayed a 9-fold enhancement in kcat/Km for the hydrolysis of the VX analog, demeton-S. An in vivo selection approach utilizing organophosphate triesters as the sole phosphorus source is discussed. The selection is based on co-expressing PTE with the phosphodiesterase (GpdQ) from E. aerogenes. Substrate specificity of GpdQ was investigated using a small library of structurally diverse organophosphate diesters and phosphonate monoesters. Results obtained from the in vivo growth assays showed that GpdQ enabled E. coli to utilize various organophosphate diesters and phosphonate monoesters as the sole phosphorus source. Cells co-expressing PTE and GpdQ were tested for their ability to utilize two different organophosphate triesters as the sole phosphorus source. The results from this experiment indicate that the growth rate is limited by the phosphotriesterase activity. Protein translocation to the periplasm was proven advantageous for in vivo selection since it overcomes the limitation of intercellular delivery of the substrate of interest. Translocation of PTE to the periplasmic space of E. coli was examined. Two signal peptides were tested; the native leader peptide from Flavobacterium sp. and the signal sequence of alkaline phosphatase. The results obtained from cellular fractionation indicated that neither signal peptides were able to translocate PTE to the periplasm and that the protein remained in the cytoplasm.Item Improving Reactivity Against Target Organothiophosphates via Active-Site Directed Mutagenisis of a Bacterial Phosphotriesterase(2013-01-17) Githens, Tyler 1986-Phosphotriesters, also known as organophosphates (OP), represent a class of toxic compounds first synthesized in Germany. Enzymatic removal of harmful insecticides and breakdown products is a promising alternative to skimming or dredging. Wild type bacterial phosphotriesterase (PTE) was screened against 7 agricultural organophosphates: coumaphos, chlorpyrifos, fenitrothion, temephos, profenofos, pirimiphosmethyl and diazinon. The initial results laid the groundwork for a mutagenesis study to investigate the determining factors in enzyme reactivity. Coumaphos is hydrolyzed more efficiently than any other target by the wild type cobalt enzyme (kcat/Km = 2 x 10^7 M^-1s^-1). Coumaphos, fenitrothion and chlorpyrifos had the lowest Km values from the initial screen and were targets for steady state kinetic characterization of active site mutants. Site directed mutagenesis of binding sites was conducted and the most reactive point mutants, F132G, F132V and S308G, were used as backgrounds for subsequent mutation. Seven active site double mutants: F132G/S308G, F132G/S308T, F132V/S308G, F132V/S308T, F132G/I106T, F132V/I106T and G308/W309 were purified to homogeneity for kinetic characterization. The double mutant G308/F132V enhanced chlorpyrifos reactivity relative to the wild type enzyme. This enhancement of reactivity is proposed to result from conformational rearrangement following substrate bond hydrolysis.Item Pesticide impact on non-target wildlife in irrigated crops: simulated impact of cholinesterase-inhibiting pesticides on white-winged doves in the Lower Rio Grande Valley of Texas(Texas A&M University, 2007-09-17) Pisani, Jorge MarceloI present a simulation model that should be a useful tool for risk assessment of the impact of insecticide inhibitors of cholinesterase (ChE) applied in irrigated agricultural fields on non-target wildlife. I developed the model as a compartment model based on difference equations (????t = 1 hour) and programmed with Stella???? VII software. Conceptually the model is compartmentalized into six submodels describing the dynamics of (1) insecticide application, (2) insecticide movement into floodable soil, (3) irrigation and rain, (4) insecticide dissolution in water, (5) foraging and insecticide intake from water, and (6) ChE inhibition and recovery. To demonstrate application of the model, I simulate historical, current, and ??????worst-case?????? scenarios, that examined the impact of ChE-inhibiting insecticides on white-winged doves (WWDO - Zenaida asiatica) in the Lower Rio Grande Valley of Texas (LRGV), USA. To my knowledge, there are no field data verifying that the cause of ChE deprivation in WWDO is due to the ingestion of ChE-inhibiting insecticide residues dissolved in drinking water. I parameterized the model to represent a system composed of fields of cotton, sorghum, corn, citrus, and brushland that encompasses the activity range of a WWDO in the LRGV. I simulated situations representing the typical scenario of WWDO using irrigated crop fields in the absence and in the presence of rain. I also simulated ??????worst case?????? scenarios in which methyl parathion was applied at high rates and high frequency. Based on results of the simulations, I conclude that it is unlikely that WWDO are seriously exposed to ChE-inhibiting insecticides by drinking contaminated water. Only in rare cases, for example, when a rain event occurs just after the application of insecticides, are levels of ChE inhibition likely to approach diagnostic levels (20 %). The present simulation model should be a useful tool to predict the effect of ChE-inhibiting insecticides on the ChE activity of different species that drink contaminated water from irrigated agricultural fields. It should be particularly useful in identifying specific situations in which the juxtaposition of environmental conditions and management schemes could result in a high risk to non-target wildlife.