Mutagenesis Of Organophosphorus-Degrading Enzymes To Enhance Hydrolysis Of DFP And The Mechanism For Catalysis Activation

Organophosphorus hydrolase(OPH),the most studied and important enzyme to degrade organophosphate compounds(OPs),has been widely used in bioremediation of OP contaminations.There has been increasing attention to ehhance OPH activity towards highly toxic OPs,including G-type nerve agents and diisopropylfluorophosphate(DFP).In this study,the genes encoding OP-degrading enzymes were cloned from bacterial genomes and expressed in E.coli BL21(DE3)as recombinant proteins.The desirable recombinant organophosphorus hydrolases(rOPHm and rMPDm)with high activities towards DFP were achieved by site-directed mutagenesis.The effects of aminoalcohols,amines,and alcohols on OPH-catalyzed hydrolysis of DFP were also investigated.The obtained results were listed below:1.The mpd and oph genes were cloned from total genomic DNA of Pseudomonas stutzeri HS-D36 and Flavobacterium sp.ATCC 27551 by PCR amplification.The length of oph was 1014 bp,encoding 337 amino acids;while mpd was 996 bp long and encoded 331 amino acids.Recombinant plasmid pET-oph and pET-mpd were constructed and heterologously expressed in E.coli BL21(DE3).SDS-PAGE indicated the molecular weight of the rOPH and rMPD were 37 and 36 KDa,respectively,and the optimum induction conditions for these two recombinant proteins were 18?/1.0 mM IPTG and 37?/1.0 mM IPTG,respectively.2.Homology modeling of OPH and MPD was conducted with wild-type OPH(PDB:ldpm)and MPD(PDB:lp9e)as templates.The three-dimensional structures showed that OPH and MPD were both homodimer with a binuclear metal center in their active sites.The OPH monomer was a(a-(3)8 fold with two Zn2+ in the enzyme metal center,and the MPD monomer was a typical(3-lactamase fold with a Zn2+/Cd2+ metal center.The simulated protein structure also suggested the interaction of active site with certain hydrophobic OP substrate(s)through leaving group pocket and the other two ester group pockets on the enzyme surface.The leaving group pocket was made up by residues W102,F103,F277 and Y280 in OPH and F119,W179,and F196 in MPD.Molecular docking analysis revealed the essential role of residues F103,L111,S72 and A147 in the OPH-catalyzed hydrolysis of DFP.The docking value of F103Y with DFP was significantly increased,as the result of formation of an additional hydrogen bond between Y103 and the substrate DFP,suggesting that F103 could be a critical point to modulate OPH activity towards DFP.It could be speculated that the corresponding amino acid residues in MPD might have similar contributions to regulating DFP-hydrolysis activities.Six OPH mutants and eight MPD mutants were successfully generated by reverse PCR and overlap extension PCR.These 14 mutants were all solubly expressed in E.coli BL21(DE3).The obtained rOPHm and rMPDm exhibited higher DFP-degrading activities without exception,supporting our speculation from molecular modeling and docking experiments that the residue F103 in OPH and residues F119,W179 and F196 in MPD were essential in the activity modulation.3.The purified rOPH and rMPD and the related mutants were used to investigate the effects of aminoalcohols,amines,and alcohols on the activity of OPH and MPD in DFP or MP hydrolysis.The results suggested that aminoalcohols could activate OPH and the orders for the activation efficiency were triethanolamine(TEA)>diethanolamine(DEA)>ethanolamine(MEA).On the other hand,the hydrolysis of DFP catalyzed by OPH displayed Michaelis-Menten-type saturation behavior in the presence of the aminoalcohols,and the associated double-reciprocal plots also gave linear curves for the aminoalcohol-activated OPH reactions.The activation folds were gradually increased with the increasing TEA concentrations in the range of 0-400 mM.The TEA-dose-dependent activation of OPH was also kinetically fit to the Michaelis-Menten equation and the associated kinetic parameters of OPH,including kcat,Vm,Km and kcat/Km,were increased with the increasing TEA concentrations.In contrast,the amines and alcohols investigated had much less or no ability to activate OPH.Inhibition studies on the hydrolysis of DFP were performed using sodium fluoride(NaF)as the inhibitor against the OPH with Zn2+ as the active-site metal.The results indicated a relatively poor affinity of DFP to OPH in the presence of fluoride.The adding of TEA to OPH buffers significantly increased the Vm and kcat/Km values at inhibitor treatment.In the presence of 300 mM TEA,the Vm and Kcat/Km at 3 mM NaF remained at 106.1± 1.1 min-1 and 36.23 ± 5.07 mM-1 s-1,respectively,approximately 4.5-folds higher than that in the absence of TEA,revealing the ability of this aminoalcohol to reduce the NaF inhibition in the OPH catalysis.In addition,the calculated Ki values for NaF in the presence of O,1 00,200,and 300 mM TEA were 3.21,5.17,5.45 and 7.51 mM,respectively.The Ki(NaF)for the OPH-catalyzed hydrolysis of DFP was increased with the increasing TEA concentrations,kinetically suggesting the reduction of fluoride inhibition by TEA.The kinetic parameters Kcat,Vm,Km and kcat/Km of OPH/MPD in the enzyme-catalyzed hydrolysis of MP/PO were relatively stable in the presense of TEA,indicating that aminoalcohols could not activate OPH/MPD in the hydrolysis of MP/PO.The result suggested that the hydrolysis of P-F bond,rather than P-O bond,could be activated by aminoalcohols.The activation effect of TEA on the OPH mutants F103Y and L111Y were much higher than that on F103A and L111A.The similar results were obtained in the analysis of MPD mutants.The results further indicated that the activation effect of aminoalcohols was dependent on the particular residues involved in the releasing of product from OPH/MPD active center.