Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Functional Characterization of the ${\alpha}$- and ${\beta}$-Subunits of a Group II Chaperonin from Aeropyrum pernix K1

  • Lee, Jin-Woo (Department of Biomaterial Control (BK21 Program), Dong-Eui University Graduate School) ;
  • Kim, Se Won (Department of Biomaterial Control (BK21 Program), Dong-Eui University Graduate School) ;
  • Kim, Jeong-Hwan (Department of Biomaterial Control (BK21 Program), Dong-Eui University Graduate School) ;
  • Jeon, Sung-Jong (Department of Biomaterial Control (BK21 Program), Dong-Eui University Graduate School) ;
  • Kwon, Hyun-Ju (Department of Biomaterial Control (BK21 Program), Dong-Eui University Graduate School) ;
  • Kim, Byung-Woo (Department of Biomaterial Control (BK21 Program), Dong-Eui University Graduate School) ;
  • Nam, Soo-Wan (Department of Biomaterial Control (BK21 Program), Dong-Eui University Graduate School)
  • Received : 2012.12.20
  • Accepted : 2013.02.03
  • Published : 2013.06.28
Download PDF
⟨ Previous Next ⟩

Abstract

We isolated and functionally characterized the ${\alpha}$- and ${\beta}$-subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d-ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at $80^{\circ}C$. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

Keywords

References

  1. Braig, K., Z. Otwinowski, R. Hegde, D. C. Boisvert, A. Joachimiak, A. L. Horwich, and P. B. Sigler. 1994. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature 371: 578-586. https://doi.org/10.1038/371578a0
  2. Buchner, J., M. Schmidt, M. Fuchs, R. Jaenicke, R. Ru-dolph, F. X. Schmid, and T. Kiefhaber. 1991. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30: 1586-1591. https://doi.org/10.1021/bi00220a020
  3. Bukau, B. and A. L. Horwich. 1998. The Hsp70 and Hsp60 chaperone machines. Cell 92: 351-366. https://doi.org/10.1016/S0092-8674(00)80928-9
  4. Chang, Z., T. P. Primm, J. Jakana, I. H. Lee, I. Sery-sheva, W. Chiu, et al. 1996. Mycobacterium tuberculosis 16-kDa antigen (Hsp 16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation. J. Biol. Chem. 271: 7218-7223. https://doi.org/10.1074/jbc.271.12.7218
  5. Chen, H. Y., Z. M. Chu, Y. H. Ma, Y. Zhang, and S. L. Yang. 2007. Expression and characterization of the chaperonin molecular machine from the hyperthermophilic archaeon Pyrococcus furiosus. J. Basic Microbiol. 47: 132-137. https://doi.org/10.1002/jobm.200610215
  6. Ellis, R. J. 1996. The Chaperonins. Academic Press, San Diego, USA.
  7. Furutani, M., T. Iida, T. Yoshida, and T. Maruyama. 1998. Group II chaperonin in a thermophilic methanogen Methanococcus thermolithotrophicus. Chaperone activity and filament-forming ability. J. Biol. Chem. 273: 28399-28407. https://doi.org/10.1074/jbc.273.43.28399
  8. Guaqliardi, A., L. Cerchia, S. Bartolucci, and M. Rossi. 1994. The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro. Protein Sci. 3: 1436-1443. https://doi.org/10.1002/pro.5560030910
  9. Gutsche, I., L. O. Essen, and W. Baumeister. 1999. Group II chaperonins: New TRic(k)s and turns of a protein folding machine. J. Mol. Biol. 293: 295-312. https://doi.org/10.1006/jmbi.1999.3008
  10. Gutsche, I., O. Mihalache, and W. Baumeister. 2000. ATPase cycle of an archaeal chaperonin. J. Mol. Biol. 300: 187-196. https://doi.org/10.1006/jmbi.2000.3833
  11. Hartl, F. U. and M. Hayer-Hartl. 2002. Molecular chaperones in the cytosol: From nascent chain to folded protein. Science 295: 1852-1858. https://doi.org/10.1126/science.1068408
  12. Hirai, H., K. Noi, K. Hongo, T. Mizobata, and Y. Kawata. 2008. Functional characterization of the recombinant group II chaperonin alpha from Thermoplasma acidophilum. J. Biochem. 143: 505-515.
  13. Isumi, M., S. Fuiwara, M. Takagi, S. Kanaya, and T. Imanaka. 1999. Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: Analysis of an ATPase-deficient mutant enzyme. Appl. Environ. Microbiol. 65: 1801-1805.
  14. Kawarabayasi, Y., Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, et al. 1999. Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1. DNA Res. 6: 83-101, 145-152. https://doi.org/10.1093/dnares/6.2.83
  15. Kim, H. and I. H. Kim. 2005. Refolding of fusion ferritin by gel filtration chromatography (GFC). Biotechnol. Bioprocess Eng. 10: 500-504. https://doi.org/10.1007/BF02932284
  16. Kim, S., K. R. Willison, and A. L. Horwich. 1994. Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains. Trends Biochem. Sci. 19: 543-548. https://doi.org/10.1016/0968-0004(94)90058-2
  17. Kohda, J., H. Kawanishi, K. Suehara, Y. Nakano, and T. Yano. 2006. Stabilization of free and immobilized enzymes using hyperthermophilic chaperonin. J. Biosci. Bioeng. 101: 131-136. https://doi.org/10.1263/jbb.101.131
  18. Kubota, H., G. Hynes, and K. Willison. 1995. The chaperonin containing t-complex polypeptide 1 (TCP-1). Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. Eur. J. Biochem. 230: 3-16. https://doi.org/10.1111/j.1432-1033.1995.tb20527.x
  19. Marco, S., D. Urena, J. L. Carrascosa, T. Waldmann, J. Peters, R. Hegerl, et al. 1994. The molecular chaperone TF55. Assessment of symmetry. FEBS Lett. 341: 152-155. https://doi.org/10.1016/0014-5793(94)80447-8
  20. Mayhew, M., A. C. da Silva, J. Martin, H. Erdjument-Bromage, P. Tempst, and F. U. Hartl. 1996. Protein folding in the central cavity of the GroEL-GroES chaperonin complex. Nature 379: 420-426. https://doi.org/10.1038/379420a0
  21. Minuth, T., G. Frey, P. Lindner, R. Rachel, K. O. Stetter, and R. Jaenicke. 1998. Recombinant homo- and heterooligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro. Eur. J. Biochem. 258: 837-845. https://doi.org/10.1046/j.1432-1327.1998.2580837.x
  22. Minuth, T., M. Henn, K. Rutkat, S. Andra, G. Frey, R. Rachel, et al. 1999. The recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri: In vitro analysis of its chaperone activity. Biol. Chem. 380: 55-62.
  23. Okochi, M., H. Matsuzaki, T. Nomura, N. Ishii, and M. Yohda. 2005. Molecular characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3. Extremophiles 9: 127-134. https://doi.org/10.1007/s00792-004-0427-y
  24. Ranson, N. A., H. E. White, and H. R. Saibil. 1998. Chaperonins. Biochem. J. 333: 233-242. https://doi.org/10.1042/bj3330233
  25. Shomura, Y., T. Yoshida, R. Iizuka, T. Maruyama, M. Yohda, and K. Miki. 2004. Crystal structures of the group II chaperonin from Thermococcus strain KS-1: Steric hindrance by the substituted amino acid, and inter-subunit rearrangement between two crystal forms. J. Mol. Biol. 335: 1265-1278. https://doi.org/10.1016/j.jmb.2003.11.028
  26. Son, H. J., E. J. Shin, S. W. Nam, D. E. Kim, and S. J. Jeon. 2007. Properties of the $\alpha$ subunit of a chaperonin from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. FEMS Microbiol. Lett. 266: 103-109. https://doi.org/10.1111/j.1574-6968.2006.00513.x
  27. Trent, J. D., E. Nimmesgern, J. S. Wall, F. U. Hartl, and A. L. Horwich. 1991. A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature 354: 490-493. https://doi.org/10.1038/354490a0
  28. Waldmann, T., E. Nimmesgern, M. Nitsch, J. Peters, G. Pfeifer, S. Muller, et al. 1995. The thermosome of Thermoplasma acidophilum and its relationship to the eukaryotic chaperonin TRiC. Eur. J. Biochem. 227: 848-856. https://doi.org/10.1111/j.1432-1033.1995.tb20210.x
  29. Wiech, H., J. Buchner, R. Zimmermann, and U. Jakob. 1992. Hsp90 chaperones protein folding in vitro. Nature 358: 169-170. https://doi.org/10.1038/358169a0
  30. Yan, Z., S. Fujiwara, K. Kohda, M. Takagi, and T. Imanaka. 1997. In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1. Appl. Environ. Microbiol. 63: 785-789.
  31. Yoshida, T., M. Yohda, T. Iida, T. Maruyama, H. Taguchi, K. Yazaki, et al. 1997. Structural and functional characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the hyperthermophilic archaeum Thermococcus strain KS-1. J. Mol. Biol. 273: 635-645. https://doi.org/10.1006/jmbi.1997.1337
  32. Zhi, W., P. Srere, and C. T. Evans. 1991. Conformational stability of pig citrate synthase and some active site mutants. Biochemistry 30: 9281-9286. https://doi.org/10.1021/bi00102a021
  33. Zhi, W., S. J. Landry, L. M. Gierasch, and P. A. Srere. 1992. Renaturation of citrate synthase: Influence of denaturant and folding assistants. Protein Sci. 1: 522-529.

Cited by

  1. Coexpression of Alginate Lyase with Hyperthermophilic Archaea Chaperonin in E. coli vol.25, pp.2, 2015, https://doi.org/10.5352/jls.2015.25.2.130