Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures

Abstract

Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application for decontamination of organophosphorus compounds in certain pesticides and chemical warfare agents under harsh conditions. However, current applications that use an enzyme-based cocktail are limited by poor long-term enzyme stability and low reactivity over a broad range of temperatures. To obtain a better enzyme for OP nerve agent decontamination and to investigate structural factors that influence protein thermostability and thermoactivity, randomly mutated P. furiosus prolidases were prepared by using XL1-red-based mutagenesis and error-prone PCR. An Escherichia coli strain JD1 (lambdaDE3) (auxotrophic for proline [DeltaproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P. furiosus prolidase expression plasmids. JD1 (lambdaDE3) cells were transformed with mutated prolidase expression plasmids and plated on minimal media supplemented with 50 muM Leu-Pro as the only source of proline. By using this positive selection, Pyrococcus prolidase mutants with improved activity over a broader range of temperatures were isolated. The activities of the mutants over a broad temperature range were measured for both Xaa-Pro dipeptides and OP nerve agents, and the thermoactivity and thermostability of the mutants were determined.

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

“…The E. coli K-12 derivative NK5525 ( proA ::Tn 10 ) was used to construct the selection strain JD1( λ DE3) as described in [20] for screening of cold-adapted P. horikoshii prolidase variants. The P. horikoshii prolidase expression plasmid pET- Ph1 prol was previously constructed as described in [16].…”

“…The enzyme activity assay protocol is based on a method previously described by [17, 20]. The reaction mixture (500  μ L) contained 50 mM MOPS buffer (3-[ N -morpholino] propanesulfonic acid) pH 7.0, 200 mM NaCl, water, 5% (vol/vol) glycerol, 100  μ g/mL BSA (bovine serum albumin) protein, 0.2 mM CoCl 2 , and the enzyme.…”

“…Prolidase samples were assayed with two additional substrates, Pro-Ala and Val-Leu-Pro, to further illustrate prolidase preference of Xaa-Pro dipeptides [20]. Kinetic parameters of the Ph1 prol mutants were determined at 70°C using a range of Leu-Pro concentrations (0.25–12 mM).…”

The Order Thermococcales and the Family Thermococcaceae

“…litoralis The Order Thermococcales and the Family Thermococcaceae . Tsunasawa et al (1998) Prolidase (proline dipeptidase) P. furiosus Ghosh et al (1998) P. horikoshii Theriot et al (2009Theriot et al ( , 2010 locations (see > Table 27.2). They are found all around the globe where hydrothermal are present (Lepage et al 2004).…”

“…(2004) The Order Thermococcales and the Family Thermococcaceae Theriot et al 2009Theriot et al , 2010Ward et al 2002b). The derived amino acids are oxidized to their corresponding organic acids with the generation of ATP by substrate-level phosphorylation.…”