After identifying D253 as a hot-spot residue, we designed the double mutant D253E-H254R and tested its effect on interactions in the OPH active site using MD simulations. We chose to mutate position 254 since His 254 influences catalysis by interacting with active site residues, particularly His 230 (Figure ). Through comparing the donor-acceptor distances in the MD trajectory of the WT OPH to the donoracceptor distances in the MD trajectory of the mutant form, we found that D253E-H254R induces three additional hydrogen bonds in the OPH active site. Out of these hydrogen bonds, two involve the side chain of Arg 254 (Figure and), which interacts with Leu 271, and His 257. The remaining hydrogen bond mediated by the double mutant occurs between Lys 169 and Asp 100 and appears after 40 ns of MD (Figure ).