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.

Prolidases hydrolyze the unique bond between X-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus compounds, including the nerve agents, soman and sarin. The advantages of using hyperthermophilic enzymes in biodetoxification strategies are based on their enzyme stability and efficiency. Therefore, it is advantageous to examine new thermostable prolidases for potential use in biotechnological applications. Two thermostable prolidase homologs, PH1149 and PH0974, were identified in the genome of Pyrococcus horikoshii based on their sequences having conserved metal binding and catalytic amino acid residues that are present in other known prolidases, such as the previously characterized Pyrococcus furiosus prolidase. These P. horikoshii prolidases were expressed recombinantly in the Escherichia coli strain BL21 (lambdaDE3), and both were shown to function as proline dipeptidases. Biochemical characterization of these prolidases shows they have higher catalytic activities over a broader pH range, higher affinity for metal and are more stable compared to P. furiosus prolidase. This study has important implications for the potential use of these enzymes in biotechnological applications and provides further information on the functional traits of hyperthermophilic proteins, specifically metalloenzymes.

Prolidases are dipeptidases specific for cleavage of Xaa-Pro dipeptides. Pyrococcus furiosus prolidase is a homodimer having one Co-bound dinuclear metal cluster per monomer with one tightly bound Co(II) site and the other loosely bound (K d 0.24 mM). To identify which Co site is tight-binding and which is loose-binding, site-directed mutagenesis was used to modify amino acid residues that participate in binding the Co1 (E-313 and H-284), the Co2 site (D-209) or the bidentate ligand (E-327). Metal-content, enzyme activity and CD-spectra analyses of D209A-, H284L-, and E327L-prolidase mutants show that Co1 is the tight-binding and Co2 the loose-binding metal center.

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Public Reporting Burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Final Report for "The Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for Optimization of Organophosphorus Nerve Agent Detoxification"Report Title ABSTRACTThe aims of this project were to structurally study and bioengineer thermostable prolidase from Pyrococcus furiosus to enable its use for oganophosphorus nerve agent detoxification. Prolidase contains one dinuclear Co metal-center/monomer and has optimal activity at 100°C, exhibiting no activity in the absence of Co2+ or at temperatures <50°C. Requirement for metal ions is characteristic of all organophosphorus nerve agent hydrolases and results from these enzymes containing dinuclear metal-centers with one tight-binding metal atom and a second loose-binding metal atom. One primary objective of this study was to determine which of the metal sites is integral and which is labile, information that will be used to bioengineer prolidases. Another objective was to produce P. furiosus prolidase mutants that have increased catalytic activity over a lower range of temperatures using random mutation and a low-temperature selection method. Three mutant prolidases targeting metal-binding amino acids have been successfully produced and biochemical analysis has demonstrated that the Co1 metal-binding site is the high-affinity site and the Co2 site, the low-affinity site. Conditions for selection of mutant prolidases with increased activity at lower temperatures have been determined and mutant prolidases (G39E and E236V) isolated that have higher activity than wild type at 37 °C.

This article unfortunately contained a mistake. Prolidase assays contained 14.8 ng for Phprol and 6.2 ng Ph1prol, MetPro (4 mM) and CoCl2 (1.2 mM). The following buffers were used for each pH at a final concentration of 100 mM: pH4.0-5.0, Sodium Acetate; pH6.0-8.0, MOPS; pH9.0, CHES; pH10.0, CAPS. 100% specific activity corresponds to 2,321 U/mg for Phprol and 3,357 U/mg for Ph1prolThe online version of the original article can be found at http://dx.doi.