Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site.

Léa El Khoury,David L Mobley,Dongmei Ye,Susan B Rempe

doi:10.3390/ijms222312624

Abstract

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.

Highlights

We hypothesized that the alterations of these residues would stabilize the Organophosphorus hydrolase (OPH) active site by increasing the hydrogen bonding associated with the side chains of the active site residues, leading to a more stable substrate binding
In contrast with previously designed OPH variants focused on modification of the active site residues to increase the catalytic activity of OPH, our current findings suggest that mutations near the enzyme active site can enhance paraoxon binding affinity
Organophosphorus hydrolase is a bacterial enzyme that can detoxify a wide range of OP nerve agents

Summary

Introduction

OP compounds cause severe neurotoxic effects by covalently binding to acetylcholinesterase (AChE), an enzyme that catalyzes the breakdown of neurotransmitters such as acetylcholine. Current medical countermeasures, including atropine and oxime-based reactivators [2] that target the down-stream pathways of OP, act through reactivation of AChE, blocking acetylcholine receptor activity or easing the symptoms. A direct method that can hydrolyze OP agents before they enter the central nervous system is needed. OPH is the only enzyme used in organophosphate remediation [3], but more investigation is needed to improve the use of OPH in medical therapy. The advantage of using OPH therapy over oxime-based treatment is rapid hydrolysis of OP agents in the circulatory system. Nerve agents are eliminated before penetrating the blood-brain barrier and exerting effects in the nervous system

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Conclusion