Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase 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. 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？–100°C 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 [3–7]. 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
References
[1]
J. M. González, Y. Masuchi, F. T. Robb et al., “Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough,” Extremophiles, vol. 2, no. 2, pp. 123–130, 1998.
[2]
G. Fiala and K. O. Stetter, “Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C,” Archives of Microbiology, vol. 145, no. 1, pp. 56–61, 1986.
[3]
M. W. W. Adams, “Enzymes and proteins from organisms that grow near and above 100°C,” Annual Review of Microbiology, vol. 47, pp. 627–658, 1993.
[4]
J. Synowiecki, B. Grzybowska, and A. Zdzieb？o, “Sources, properties and suitability of new thermostable enzymes in food processing,” Critical Reviews in Food Science and Nutrition, vol. 46, no. 3, pp. 197–205, 2006.
[5]
S. E. Blumer-Schuette, I. Kataeva, J. Westpheling, M. W. Adams, and R. M. Kelly, “Extremely thermophilic microorganisms for biomass conversion: status and prospects,” Current Opinion in Biotechnology, vol. 19, no. 3, pp. 210–217, 2008.
[6]
L. D. Unsworth, J. Van Der Oost, and S. Koutsopoulos, “Hyperthermophilic enzymes—stability, activity and implementation strategies for high temperature applications,” The FEBS Journal, vol. 274, no. 16, pp. 4044–4056, 2007.
[7]
K. Egorova and G. Antranikian, “Industrial relevance of thermophilic Archaea,” Current Opinion in Microbiology, vol. 8, no. 6, pp. 649–655, 2005.
[8]
C. M. Theriot, S. R. Tove, and A. M. Grunden, “Biotechnological applications of recombinant microbial prolidases,” in Advances in Applied Microbiology, A. Laskin, G. Gadd, and S. Sariaslani, Eds., vol. 68, pp. 99–132, Academic Press, New York, NY, USA, 2009.
[9]
W. T. Lowther and B. W. Matthews, “Metalloaminopeptidases: common functional themes in disparate structural surroundings,” Chemical Reviews, vol. 102, no. 12, pp. 4581–4607, 2002.
[10]
T. C. Cheng, J. J. DeFrank, and V. K. Rastogi, “Alteromonas prolidase for organophosphorus G-agent decontamination,” Chemico-Biological Interactions, vol. 119-120, pp. 455–462, 1999.
[11]
T. C. Cheng, V. K. Rastogi, J. J. Defrank, and G. P. Sawiris, “G-type nerve agent decontamination by Alteromonas prolidase,” Annals of the New York Academy of Sciences, vol. 864, pp. 253–258, 1998.
[12]
N. K. Vyas, A. Nickitenko, V. K. Rastogi, S. S. Shah, and F. A. Quiocho, “Structural insights into the dual activities of the nerve agent degrading organophosphate anhydrolase/prolidase,” Biochemistry, vol. 49, no. 3, pp. 547–559, 2010.
[13]
C. M. Theriot and A. M. Grunden, “Hydrolysis of organophosphorus compounds by microbial enzymes,” Applied Microbiology and Biotechnology, vol. 89, pp. 35–43, 2011.
[14]
T. C. Cheng and J. J. DeFrank, “Hydrolysis of organophosphorus compounds by bacterial prolidases,” in Enzymes in Action Green Solutions for Chemical Problems, B. Zwanenburg, M. Mikolajczyk, and P. Kielbasinski, Eds., vol. 33, Kluwer Academic, Dodrecht, The Netherlands, 2000.
[15]
Joint Science and Technology Office for Chemical and Biological Defense FY 10/11-new initiatives. Defense Threat Reduction Agency, pp. 1-53, 2008.
[16]
C. M. Theriot, S. R. Tove, and A. M. Grunden, “Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii,” Applied Microbiology and Biotechnology, vol. 86, no. 1, pp. 177–188, 2010.
[17]
M. Ghosh, A. M. Grunden, D. M. Dunn, R. Weiss, and M. W. W. Adams, “Characterization of native and recombinant forms of an unusual cobalt- dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus,” Journal of Bacteriology, vol. 180, no. 18, pp. 4781–4789, 1998.
[18]
J. Jeyakanthan, et al., “Crystal structural and functional analysis of the putative dipeptidase from Pyrococcus horikoshii OT3,” Journal of Biophysics, vol. 2009, Article ID 434038, 2009.
[19]
M. J. Maher, M. Ghosh, A. M. Grunden et al., “Structure of the Prolidase from Pyrococcus furiosus,” Biochemistry, vol. 43, no. 10, pp. 2771–2783, 2004.
[20]
C. M. Theriot, X. Du, S. R. Tove, and A. M. Grunden, “Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures,” Applied Microbiology and Biotechnology, vol. 87, no. 5, pp. 1715–1726, 2010.
[21]
F. W. Studier, “Protein production by auto-induction in high density shaking cultures,” Protein Expression and Purification, vol. 41, no. 1, pp. 207–234, 2005.
[22]
C. M. Hill, W. S. Li, T. C. Cheng, J. J. DeFrank, and F. M. Raushel, “Stereochemical specificity of organophosphorus acid anhydrolase toward p-nitrophenyl analogs of soman and sarin,” Bioorganic Chemistry, vol. 29, no. 1, pp. 27–35, 2001.
