**Abstract**

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.

**Keywords:** organophosphorus hydrolase, nerve agent, paraoxon, substrate binding affinity, unnatural amino acid substitution
