Directed Evolution of Phosphotriesterase for Stereoselective Detoxification of Organophosphate Nerve Agents
The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta possess very broad substrate specificity for organophosphorus compounds. It is capable of hydrolyzing several insecticides including paraoxon and various chemical warfare agents such as sarin (GB), soman (GD), cyclosarin (GF) and VX. The catalytic ability of PTE for the hydrolysis of paraoxon is close to the limit of diffusion of the reactant in solution. However, the catalytic activity of PTE for the organophosphate nerve agents is lower than that for paraoxon. It was reported that the wild-type PTE preferentially catalyze the hydrolysis of the less toxic Rp- enantiomers of organophosphate nerve agents and their analogues than the more toxic Sp- enantiomers. The first generation of PTE mutants that contains a modified substrate binding pocket was identified and it was observed that their catalytic activity towards the more toxic Sp- enantiomers organophosphate nerve agent analogues was enhanced. The H254G/H257W/L303T mutant was shown to have a reversed stereoselectivity. The kcat/Km values of this mutant towards the hydrolysis of the SpRc- and SpSc-enantiomers of the GD analogue and the Sp-enantiomer of the GF analogue were enhanced by 73-, 543-, and 1340-fold relatively to the wile-type enzyme, respectively. The second generation of PTE mutants were isolated and shown to have higher activity toward the Sp-enantiomers of the GD and GF analogues than the first generation mutants. Saturation mutagenesis, in vitro screening and in vivo selection were conducted using the gene for the mutants from the first generation. The GWT-d3 mutant was identified as the most active PTE mutant towards the hydrolysis of the Spenantiomers of the GD analogue, the kcat/Km values were 780- and 3530-fold higher than the wild-type enzyme toward the SpRc- and SpSc-enantiomers of the GD analogues. The GWT-f5 mutant was the best PTE mutant towards the Sp-enantiomer of the GF analogue, the kcat/Km values were 15500-fold higher than the wild-type enzyme. The X-ray crystal structures of the wild-type PTE and the G60A mutant were determined in the presence of the hydrolysis product diethyl phosphate and a product analogue cacodylate, respectively. This result supports the reaction mechanism previously proposed by Dr. Sarah Aubert.