Substrate binding mechanism of glycerophosphodiesterase towards pesticides and improvement of stability by mutational analysis

Abstract

Molecular dynamics studies have been providing a great number of atomistic details of biological systems to understand enzymatic activity and underlying reaction mechanisms. Herein, molecular dynamic (MD) simulations were employed to study the binding mechanisms of organophosphate pesticides towards two metallohydrolases, i.e., glycerophosphodiesterase (GpdQ) and methyl parathion hydrolase (MPH). OPH are the class of enzymes which breaks down harmful organophosphates, including the pesticides and warfare agents into less harmful byproducts. Hence, these enzymes are excellent candidates for bioremediation. In the present study, various nonspecific organophosphate pesticides were docked into GpdQ and MPH enzymes to construct the pesticide/protein complex for performing MD simulations for 500 ns. In the first part, profenofos, chlorpyrifos and diazinon in complex with GpdQ were carried out. From MD results, all three were well stabilized by the active site and the residues P228, Q166, M167, I170, Y230. However, the distance between the metal ions was increased to accommodate the bulkier substrates. In part II, methyl paraxon, dichlorvos and profenofos binding to the cobalt and zinc bound MPH enzyme were studied. It was observed that methyl paraxon and dichlorvos favored cobalt bound MPH, whereas the profenofos had better binding free affinity towards zinc bound MPH. All pesticides coordinated with beta metal ion with a distance of 1.90 – 1.98 Å. In part III, the inhibition activity of cobalt bound MPH by carbamates such as carbofuran and carbaryl was investigated by in vitro and in silico studies. Enzyme kinetics study and Lineweaver-Burk plots exhibited uncompetitive inhibition for both carbamates. The molecular docking and MD simulations showed that among three possible binding sites, both carbamates preferred to bind at the groove between the two chains. The residues A260, P322 and L303 stabilized through π-alkyl interactions and H-bonds with S87, R319, and F320 were formed. This study has provided promising results of effective use of the organophosphate hydrolases in bioremediation.