Improving the Catalytic Activity of HyperthermophilicPyrococcus horikoshiiProlidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin. Ph1 prol (PH0974) has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pf prol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1 prol enzymes were prepared. Four Ph1 prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V- Ph1 prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases.