[en] The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified. fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Siddiqui, K. S.
Feller, Georges ; Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie
Aghajari, N., G. Feller, C. Gerday, and R. Haser. 1998. Crystal structure of the psychrophilic α-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci. 7:564-572.
Cavicchioli, R., and K. S. Siddiqui. 2004. Cold-adapted enzymes, p. 615-638. In A. Pandey, C. Webb, C. R. Soccol, and C. Larroche (ed.), Enzyme technology. AsiaTech Publishers, New Delhi, India.
Cavicchíoli, R., K. S. Siddiqui, D. Andrews, and K. R. Sowers. 2002. Low-temperature extremophiles and their applications. Curr. Opin. Biotechnol. 13:253-261.
Creighton, T. E., and R. H. Paine. 1980. Unfolding and refolding of Staphylococcus aureus penicillinase by urea-gradient electrophoresis. J. Mol. Biol. 137:431-436.
D'Amico, S., P. Claverie, C. Collins, D. Georlette, E. Gratia, A. Hoyoux, M.-A. Meuwis, G. Feller, and C. Gerday. 2002. Molecular basis of cold adaptation. Philos. Trans. R. Soc. Lond. B 357:917-925.
D'Amíco, S., C. Gerday, and G. Feller. 2001. Structural determinants of cold adaptation and stability in a large protein. J. Biol. Chem. 276:25791-25796.
D'Amico, S., C. Gerday, and G. Feller. 2003. Temperature adaptation of proteins: engineering mesophilic-like activity and stability in a cold-adapted α-amylase. J. Mol. Biol. 332:981-988.
D'Amico, S., J. C. Marx, C. Gerday, and G. Feller. 2003. Activity-stability relationship in extremophilic enzymes. J. Biol. Chem. 278:7891-7896.
Evans, R. W., and J. Williams. 1980. The electrophoresis of transferrins in urea/polyacrylamide gels. Biochem. J. 189:541-546.
Feller, G. 2003. Molecular adaptations to cold in psychrophilic enzymes. Cell. Mol. Life Sci. 60:648-662.
Feller, G., and C. Gerday. 2003. Psychrophilic enzymes: hot topics in cold adaptation. Nat. Rev. Microbiol. 1:200-208.
Feller, G., D. D'Amico, and C. Gerday. 1999. Thermodynamic stability of a cold-active α-amylase from Antarctic bacterium Alteromonas haloplanctis. Biochemistry 38:4613-4619.
Feller, G., F. Payan, F. Theys, M. Qian, R. Hasser, and C. Gerday. 1994. Stability and structural analysis of α-amylase from the Antarctic psychrophilc Alteromonas haloplanctis A23. Eur. J. Biochem. 222:441-447.
Georlette, D., V. Blaise, T. Collins, S. D'Amico, E. Gratia, A. Hoyoux, J.-C. Marx, G. Sonan, G. Feller, and C. Gerday. 2004. Some like it cold: biocatalysis at low temperatures. FEMS Microbiol. Rev. 28:25-42.
Goldenberg, D. P. 1989. Analysis of protein conformation by gel electrophoresis, p. 225-250. In T. E. Creighton (ed.), Protein structure: a practical approach. IRL Press, Oxford, United Kingdom.
Goldenberg, D. P., and T. E. Creighton. 1984. Gcl electrophoresis in studies of protein conformation and folding. Anal. Biochem. 138:1-18.
Kashiwagi, K., K. Shiba, K. Fukami-Kobayashi, T. Noda, K. Nishikawa, and H. Noguchi. 2003. Characterization of folding pathways of the type-1 and type-2 periplasmic binding proteins MgIB and ArgT. J. Biochem. 133:371-376.
Kumar, S., J.-G. Tsai, and R. Nussinov. 2002. Maximal stabilities of reversible two-state proteins. Biochemistry 41:5359-5374.
Lonhienne, T., C. Gerday, and G. Feller. 2000. Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim. Biophys. Acta 1543:1-10.
Makhtadze, G. I., and P. L. Privalov. 1994. Hydration effects in protein unfolding. Biophys. Chem. 51:291-309.
Mast, A. E., J. J. Enghild, S. V. Pizzo, and G. Salvesen. 1991. Analysis of the plasma elimination kinetics and conformational stabilities of native, proteinase-complexed, and reactive site cleaved serpins: comparison of α2-proteinase inhibitor, α1-antichymotrypsin, antithrombin III, α2-antiplasmin, angiotensinogen, and ovalbumin. Biochemistry 30:1723-1730.
Saunders, N., T. Thomas, P. M. G. Curmi, J. S. Mattick, E. Kuczek, R. Slade, J. Davis, P. D. Franzmann, D. Boone, K. Rusterholtz, R. Feldman, C. Gates, S. Bench, K. Sowers, K. Kadner, A. Aerts, P. Dehal, C. Detter, T. Glavina, S, Lucas, P. Richardson, F. Larimer, L. Hauser, M. Land, and R. Cavicchioli. 2003. Mechanisms of thermal adaptation revealed from the genomes of the Antarctic Archaea, Methanogenium frigidum and Methanococcoides burtonii. Genome Res. 13:1580-1588.
Siddiqui, K. S. 1990. Proteolytic nicking and carboxyl group modification of Arthrobacter D-xylose isomerase. Ph.D. thesis. London University, London, United Kingdom.
Siddiqui, K. S., M. Rangarajan, B. S. Hartley, A. Kitmitto, M. Panico, I. P. Blench, and H. R. Morris. 1993. Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin. Biochem. J. 289:201-208.
Uversky, V. N. 2002. Cracking the folding code. Why do some proteins adopt partially folded conformations, whereas others don't? FEBS Lett. 514:181-183.
Uversky, V. N., J. R. Gillespie, and A. J. Fink. 2000. Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins 41:415-427.
Yamashita, H., T. Nakatsuka, and M. Hirose. 1995. Structural and functional characteristics of partially disulfide-reduced intermediates of ovotransferrin N lobe. J. Biol. Chem. 270:29806-29812.
