Organophosphosphorus (OP) compounds are potent neurotoxins that inhibit acetylcholinesterase (AChE). With AChE inhibited, there is an excess of acetylcholine (ACh) molecules that continually bind to the receptors and over stimulate subsequent pathways, which can eventually lead to loss of nervous and other physiological functions. In agriculture, OPs are extensively applied as insecticides and thus bring increasing concerns of health and environmental risks. Among the OPs used in agricultural industries, chlorpyrifos (CPF) is widespread; in fact, the Environmental Protection Agency has estimated over 30% of crop producing farms currently use CPF for pest control in the United States. Therefore, efficient detoxification of CPF has become a considerable interest and an essential matter. Our research team has been developing phosphotriesterase (PTE) from Pseudomonas diminuta to improve its stability and hydrolysis of several OPs. Recently, we have demonstrated that the residue-specific incorporation of the non-canonical amino acid, para-fluorophenylalanine (pFF) into PTE (pFF-PTE) resulted in enhanced stability. Using pFF-PTE as a foundation, we employ multiple modeling methods including Rosetta©, Autodock©, molecular dynamics (MD) simulations, and quantum mechanical (QM) simulations to design variants that detoxify CPF with greater efficiency. Initially, CPF was docked in the catalytic site of PTE to identify substrate orientations consistent with the theoretical reaction mechanism for CPF hydrolysis. QM simulations were then used to generate transition state structures for CPF hydrolysis. 15 residues surrounding both unreacted and transition state CPF excluding essential divalent cation coordinating residues were algorithmically varied using Rosetta to improve binding to the PTE active site as compared to the mean ligand binding score of unmutated PTE and pFF-PTE. MD trajectories were generated for those designed structures with improved CPF binding. The candidate structures that best maintained the ideal geometry for CPF hydrolysis over the course of the trajectory were selected for synthesis and assay followed by analysis using Michaelis-Menten kinetics.

Computationally designed fluorinated phosphotriesterases for detoxification of chlorpyrifos

254th National Meeting (2017)

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