RESUMEN
Through the use of molecular and biochemical experiments and bioinformatic tools, this work demonstrates that the PA4921 gene of the Pseudomonas aeruginosa PAO1 genome is a gene responsible for cholinesterase (ChoE) activity. Similar to the acetylcholinesterase (AchE) of Zea mays, this ChoE belongs to the SGNH hydrolase family. In mature ChoE, i.e., without a signal peptide, (18)Ser, (78)Gly, (127)N, and (268)H are conserved aminoacyl residues. Acetylthiocholine (ATC) and propionylthiocholine (PTC) are substrates of this enzyme, but butyrylcholine is an inhibitor. The enzyme also catalyzes the hydrolysis of the artificial esters p-nitrophenyl propionate (pNPP) and p-nitrophenyl butyrate (pNPB) but with lower catalytic efficiency with respect to ATC or PTC. The second difference is that pNPP and pNPB did not produce inhibition at high substrate concentrations, as occurred with ATC and PTC. These differences plus preliminary biochemical and kinetic studies with alkylammonium compounds led us to propose that this enzyme is an acetylcholinesterase (AchE) or propionylcholinesterase. Studies performed with the purified recombinant enzyme indicated that the substrate saturation curves and the catalytic mechanism are similar to those properties described for mammalian AchEs. Therefore, the results of this work suggest that the P. aeruginosa ChoE is an AchE that may also be found in Pseudomonas fluorescens.
Asunto(s)
Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Acetilcolinesterasa/química , Acetilcolinesterasa/clasificación , Acetiltiocolina/metabolismo , Colina/análogos & derivados , Colina/metabolismo , Secuencia Conservada , Inhibidores Enzimáticos/metabolismo , Hidrolasas/química , Hidrolasas/clasificación , Hidrolasas/genética , Hidrolasas/metabolismo , Cinética , Filogenia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tiocolina/análogos & derivados , Tiocolina/metabolismo , Zea mays/enzimología , Zea mays/genéticaRESUMEN
Genetically modified acetylcholinesterase (AChE) from Drosophila melanogaster (dm) and from commercial sources, Electric eel (ee), Bovine erythrocites (be) and Human erythrocites (he), were investigated as biological receptors for the detection of methamidophos pesticide based on inhibition studies. Most engineered variant of AChE from dm showed enhanced sensitivity toward methamidophos pesticide. Among 24 dmAChE variants tested, 12 presented a sensitivity comparable to the commercially available eeAChE, but higher than AChEs from be and he. Four were found more sensitive and six others were insensitive to methamidophos insecticide. The D375G,Y370F,Y374A,F376L mutant was the most sensitive, with a ki value of 2.2 X 10(6) mol(-1) L min(-1), three orders of magnitude higher than eeAChE (1.1 X 10(3) mol(-1) L min(-1)). The sensor constructed with genetically modified enzyme showed better characteristics with respect to detection limit and sensitivity compared with those using commercial eeAChE. Differential pulse polarography and chronoamperometry were used as electrochemical techniques to characterize the AChE biosensors. The lower detection limit of 1 ppb was obtained with D375G,Y370F,Y374A,F376L mutant of dmAChE, compared to 90 ppb for the commercial eeAChE. This study may stimulate scientists to develop more sensitive and selective procedures for organophosphorus insecticides detection by using engineered variant of dmAChE.
Asunto(s)
Acetilcolinesterasa/química , Acetilcolinesterasa/genética , Técnicas Biosensibles/instrumentación , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Monitoreo del Ambiente/instrumentación , Compuestos Organotiofosforados/análisis , Ingeniería de Proteínas/métodos , Acetilcolinesterasa/clasificación , Animales , Técnicas Biosensibles/métodos , Bovinos , Electroquímica/instrumentación , Electroquímica/métodos , Electrophorus , Monitoreo del Ambiente/métodos , Activación Enzimática , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/genética , Humanos , Plaguicidas/análisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.