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

Abstract

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. Ph1prol (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, Pfprol (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 Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) 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.

Temperature dependent mistranslation in a hyperthermophile adapts proteins to lower temperatures

“…Conversely, when assayed at 100°C, the high-fidelity lysate has 1.35-fold higher activity. Targeted mutations in recombinants proteins that compromise protein activity or stability at higher temperatures to create more flexibility and increase activity at lower temperatures have been reported for several other thermophilic enzymes ( 35 – 39 ). The lower citrate synthase activity of the high-fidelity protein at 60°C or the lower activity of the low-fidelity protein at 100°C can be either due to decreased enzyme activities or reduced level of correctly folded proteins or a combination of both.…”

“…Furthermore, while rigid hyperthermophilic proteins have reduced Met content ( 32 ), extremely flexible psychrophilic proteins—including psychrophilic citrate synthases ( 41 , 42 )—frequently have greater Met content than their mesophilic counterparts ( 42 , 43 ), which corroborates the role of Met for naturally facilitating protein flexibility ( 42 , 44 ). Moreover, there are numerous examples of destabilizing mutations in thermophilic proteins which can increase catalysis at lower temperatures by compromising stability at higher temperatures ( 35 – 39 ). Therefore, not only are Leu-to-Met substitutions an experimentally validated method to reduce protein stability, but protein destabilization is a validated method to increase enzymatic activity at lower temperatures in thermophilic proteins.…”

Enzymatic detoxification of organophosphorus pesticides and related toxicants

“…A new variant of prolidase modified from the previously known thermophilic bacteria Pyrococcus horikoshii showed 8 times higher activity for diisopropyl-phosphorofluoridate transformation when compared to wild-type P. furiosus prolidase. 115) The monooxigenase family of cytochromes P450 (CYPs) is also able to transform OPPs. The role of CYPs on the metabolism of pesticides in humans has been studied extensively.…”

Organophosphate-degrading metallohydrolases: Structure and function of potent catalysts for applications in bioremediation

“…to have low levels of OP-degrading activity that could be improved using directed evolution [77]. For example, the prolidase from Pseudomonas horikoshii (Phprol) has been the subject of in vitro evolution experiments in which a Ph1prol gene library was constructed and screened for mutants that show increased prolidase activity at 30 • C. Four mutants were found with improved catalytic parameters at various temperatures for the hydrolysis of several OP nerve agents, including p-nitrophenol Soman [41,78].…”