%0 Journal Article
%T Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures
%A Casey M. Theriot
%A Rebecca L. Semcer
%A Saumil S. Shah
%A Amy M. Grunden
%J Archaea
%D 2011
%I Hindawi Publishing Corporation
%R 10.1155/2011/565127
%X 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. 1. Introduction Pyrococcus horikoshii and Pyrococcus furiosus are both hyperthermophilic archaea, growing optimally at 98£¿¨C100¡ãC that were isolated from a deep hydrothermal vent in the Okinawa Trough in the northeastern Pacific Ocean and from a shallow marine solfatara at Vulcano Island off the coast of Italy,respectively [1, 2]. Pyrococcus spp. are some of the most studied hyperthermophilic archaea to date owing in part to their utility for a variety of biotechnological applications [3¨C7]. For example, recombinant prolidases from Pyrococcus spp. are being studied for their potential use in bio-decontamination applications [8]. Prolidases function in vivo to hydrolyze dipeptides with proline in the C-terminus, Xaa-Pro, and a non-polar amino acid in the N-terminus [9]. However, studies have demonstrated that prolidases can also hydrolyze and detoxify organophosphate (OP) compounds such as chemical warfare agents (CWA) [8]. Two enzymes that have been characterized for potential field detoxification of nerve agents are organophosphorus acid anhydrolase (OPAA) and phosphotriesterase (PTE) [10, 11]. Recently, the crystal structure of OPAA from Alteromonas sp. JD6.5 strain has been solved, and it has been determined to be a prolidase [12]. While OPAA does have the capability to degrade OP nerve agents, its activity can be limited by exposure to high temperatures and solvents during use in field situations [13, 14]. In 2008, the Defense Threat Reduction Agency (DTRA), under the auspices of the Department of Defense recognized the importance of developing enzyme-based OP nerve agent detoxification systems and created an initiative calling for
%U http://www.hindawi.com/journals/archaea/2011/565127/