Improved Hydrolysis of Organophosphorus Compounds by Engineered Human Prolidases

Human prolidase is a proline-specific metallopeptidase that catalyzes the hydrolysis of dipeptides containing a proline residue at the C-terminus. Human prolidase also has weak hydrolytic activity for toxic organophosphorus compounds, including diisopropyl fluorophosphate (DFP), sarin and soman. In this study, we performed in silico analysis to improve the catalytic activity of human prolidase towards DFP hydrolysis. We predicted that single amino acid substitution mutations of either A252R or P365R could form a salt bridge between the A252R mutant residue and the E249 residue or the P365R mutant residue and the E424 residue. The predicted values for ΔGstability and ΔGaffinity due to salt bridge formation were examined: for A252R, ΔGstability = -12.8 kcal/mol and ΔGaffinity = -0.02 kcal/mol; for P365R, ΔGstability = -1.44 kcal/mol and ΔGaffinity = -2.98 kcal/mol. Next, we produced wild-type (WT), A252R, P365R and A252R/P365R recombinant human prolidases from a bacterial expression system with a purity of over 99 %. Finally, we measured the DFPase and prolidase activities of the purified recombinant human prolidases. The catalytic efficiencies of A252R and P365R towards DFP hydrolysis were 1.23- and 1.36-fold increases, respectively, compared to that of the WT, while the prolidase activities of A252R and P365R towards Leu-Pro hydrolysis were 0.88- and 0.78-fold that of the WT, respectively. However, we could not detect any enzymatic activity in the recombinant A252R/P365R double substitution mutant. Thus, our results indicate that substitution mutations of A252R and P365R in human prolidase show improved DFPase activity.