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.

Nerve agents are a group of organophosphorus (OP) compounds that are potent neurotoxins used as chemical warfare agents and insecticides. Current medical countermeasures, including atropine and oxime-based reactivators, target the down-stream pathways inhibited by OP agents and cannot effectively eliminate OP agents. In contrast, organophosphorus hydrolase (OPH) is a bacterial enzyme that can detoxify a wide range of OP agents. The advantage of OPH over oxime-based treatment is rapid hydrolysis of these agents in the circulatory system. Kinetic properties of OPH from various bacteria have been studied by others. Substrate binding affinity ranges between 200 μM and 2.5 mM, well above lethal levels. To improve OPH mutant screening capability, we optimized a cell-free protein synthesis system to express active OPH variants rapidly and conduct kinetic measurements. We also tested the hypothesis that active site mutations using only natural amino acids restrict the development of OPH variants with binding affinities close to nerve agent lethal levels (a few μM). Our work resulted in a suite of OPH variants that incorporated selected unnatural amino acids into OPH, with mutations targeted for the first time to both active and allosteric binding sites. Kinetic studies of those mutants show significantly improved OPH substrate binding affinity.