Improving The Catalytic Efficiency Of Methyl Parathion Hydrolyse By In Vitro Molecular Evolution

Organophosphates?OPs?have been used as nerve agents and pesticides due to their extreme toxicity,which have caused serious environmental and human health problems due to the inhibition of acetylcholinesterase.Methyl parathion hydrolase?MPH?that hydrolyzes a wide range of organophosphorus pesticides can be used to remediate land polluted by the pesticides.Moreover,this enzyme can be efficiently expressed in E.coli with high solubility.However,hydrolytic activity of MPH for organophosphate nerve agents has not yet reported,only degradation activity for some kinds of organophosphate pesticides was known.Therefore,it is imperative to apply biotechnology approach to develop MPH with good biological stability,broad-spectrum,and high-efficiency on organophosphorus pesticides.In this study,we combined random mutagenesis,site-saturation mutagenesis to engineer MPH.the catalytic efficiency of methyl parathion hydrolase from Pseudomonas sp.WBC-3was enhanced by searching and engineering a critical site far away from the binding pocket.Main research results are as follows:?1?In the first round,a four-site mutant D172H/D265V/S277I/K316R with a modest increased catalytic efficiency(3.2 folds k_cat/K_m value of the wild type)was obtained with random mutagenesis.By splitting and re-combining the four substitutions in the mutant,the critical site S277,was identified to show the most significant effects of improving binding affinity and catalytic efficiency.?2?With further site-saturation mutagenesis focused on the residue S277,another two substitutions were discovered to have even more significant decrease in K_m?40.2 and 47.6?M?and increased in k_cat/K_m values?9.5 and 10.3 folds of the wild type?compared to the original four-site mutant?3.0 and 3.2 folds?.In the three-dimensional structure,residue S277 is located at a hinge region of a loop,which could act as a‘lid'at the substrate entering to the binding pocket.This suggests that substitutions on residue S277 could affect substrate binding via conformational change in substrate entrance region.?3?By combing MPH crystal structure information and bioinformatics analysis,a more targeted selection of hot-spot amino acids for enzyme modification was performed.Compared to the WT,the catalytic efficiency of the mutant Q272E was increased to 5.5-fold towards methyl parathion.This work provides a valuable protocol combining random mutagenesis,site-saturation mutagenesis,structural and bioinformatics analyses to obtain mutants with high catalytic efficiency from a screening library of a modest size?3800 strains?.