**2. Bio-engineered OPH degradation of nerve agent analog: paraoxon**

#### **2.1 Methods**

#### *2.1.1 Identify OPH mutation sites with molecular simulation*

Computational studies, to identify OPH mutation sites, used chain A of the OPH crystal structure of paraoxon analog and diethyl 4-methylbenzylphosphonate, co-crystallized with OPH [5]. In contrast to most previous structural studies, which assumed that the binding orientation of paraoxon resembles the substrate analog [6–8], we identified a different binding mode for paraoxon compared to its analog diethyl 4-methylbenzylphosphonate [9]. To do this, we used three different docking algorithms (POSIT, HYBRID, and FRED) available in OPENEYE software

#### *Neurotoxin Decontamination DOI: http://dx.doi.org/10.5772/intechopen.110853*

(OEDocking 3.2.0, OpenEye Inc.) to generate fifty potential binding modes of paraoxon in the OPH binding site. After down-selecting to the two most dissimilar paraoxon-OPH docking poses, we carried out molecular dynamics (MD) simulations over long times, up to 105 ns, to obtain the most stable paraoxon binding mode and define binding interactions at the OPH active site. MD simulation was also performed to identify mutation sites that can stabilize paraoxon binding.

Details of the MD simulations can be found in Ref. [9]. In brief, we used the OpenMM simulation package 7.11. We assigned partial charges to the substrate (paraoxon) atoms using the AM1-BCC charge model [10]. Amberff14SB and GAFF vs. 1.8 force fields were applied to the protein and substrate, respectively. We applied the TIP3P force field to describe water. After solvating the protein-substrate complex in water in a cubic box of 10 Å on a side, Na<sup>+</sup> and Cl− counter ions were added to neutralize the system. We set the simulation time step to 4 fs and applied the hydrogen mass repartitioning approach [11, 12]. Long-ranged electrostatic interactions were calculated using the particle mesh Ewald approach [13], with a cut-off of 10 Å for the real-space electrostatic and Lennard-Jones forces. After minimizing the energy of the water and ions, while keeping the protein and substrate restrained using 500 kcal/ mol-Å2 positional restraints, we performed a second energy minimization step.
