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Fundamentals of Cold-Adapted Enzymes

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Psychrophiles: from Biodiversity to Biotechnology

As already discussed in the preceding chapters, life at low temperatures is abundant, diverse and widespread, with organisms from all three domains of life being represented. Bacteria and archaea at thermal equilibrium with their environment are found to be preponderant, and these cold-adapted, or psychrophilic, microorganisms have been found to not only endure, but to flourish under the harsh conditions of permanently low-temperatures. In fact, for some, this environment is not only optimal, but mandatory for sustained cell proliferation, with moderate to high temperatures (e.g., >12°C) being deleterious (Xu et al. 2003c).

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References

  • Aghajari N, Feller G, Gerday C, Haser R (1998a) Structures of the psychrophilic Alteromonas haloplanctis -amylase give insights into cold adaptation at a molecular level. Structure 6:1503–1516.

    Article  CAS  PubMed  Google Scholar 

  • Aghajari N, Feller G, Gerday C, Haser R (1998b) Crystal structures of the psychrophilic A-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci 7:564–572.

    Article  CAS  PubMed  Google Scholar 

  • Aghajari N, Van Petegem F, Villeret V, Chessa JP, Gerday C, Haser R, Van Beeumen J (2003) Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases. Proteins 50:636–647.

    Article  CAS  PubMed  Google Scholar 

  • Alvarez M, Zeelen JP, Mainfroid V, Rentier-Delrue F, Martial JA, Wyns L, Wierenga RK, Maes D (1998) Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus. Kinetic and structural properties. J Biol Chem 273:2199–2206.

    Article  CAS  PubMed  Google Scholar 

  • Arnorsdottir J, Kristjansson MM, Ficner R (2005) Crystal structure of a subtilisin-like serine proteinase from a psychrotrophic Vibrio species reveals structural aspects of cold adaptation. Febs J 272:832–845.

    Article  CAS  PubMed  Google Scholar 

  • Arrhenius S (1889) Uber die Reaktionsgeschwindigkeit bei der Inversion von Rohrzucker durch Säuren. Z Physik Chem 4:226–248.

    Google Scholar 

  • Bae E, Phillips GN Jr (2004) Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases. J Biol Chem 279:28202–28208.

    Article  CAS  PubMed  Google Scholar 

  • Bendt A, Huller H, Kammel U, Helmke E, Schweder T (2001) Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7. Extremophiles 5:119–126.

    Article  CAS  PubMed  Google Scholar 

  • Bentahir M, Feller G, Aittaleb M, Lamotte-Brasseur J, Himri T, Chessa JP, Gerday C (2000) Structural, kinetic, and calorimetric characterization of the cold-active phosphoglycerate kinase from the antarctic Pseudomonas sp. TACII18. J Biol Chem 275:11147–11153.

    Article  CAS  PubMed  Google Scholar 

  • Berglund GI, Willassen NP, Hordvik A, Smalas AO (1995) Structure of native pancreatic elastase from North Atlantic salmon at 1.61 A resolution. Acta Crystallogr D Biol Crystallogr 51:925–937.

    Article  CAS  PubMed  Google Scholar 

  • Birolo L, Tutino ML, Fontanella B, Gerday C, Mainolfi K, Pascarella S, Sannia G, Vinci F, Marino G (2000) Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125. Cloning, expression, properties, and molecular modelling. Eur J Biochem 267:2790–2802.

    Article  CAS  PubMed  Google Scholar 

  • Brandsdal BO, Heimstad ES, Sylte I, Smalas AO (1999) Comparative molecular dynamics of mesophilic and psychrophilic protein homologues studied by 1.2 ns simulations. J Biomol Struct Dyn 17:493–506.

    CAS  PubMed  Google Scholar 

  • Chessa JP, Petrescu I, Bentahir M, Van Beeumen J, Gerday C (2000) Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species. Biochim Biophys Acta. 1479:265–274.

    CAS  PubMed  Google Scholar 

  • Claverie P, Vigano C, Ruysschaert JM, Gerday C, Feller G (2003) The precursor of a psychrophilic alpha-amylase: structural characterization and insights into cold adaptation. Biochim Biophys Acta 1649:119–122.

    CAS  PubMed  Google Scholar 

  • Collins T, Claverie P, D’Amico S, Georlette D, Gratia E, Hoyoux A, Meuwis MA, Poncin J, Sonan G, Feller G, Gerday C (2002a) Life in the cold: psychrophilic enzymes. In: Pandalai SG (ed.), Recent Research Developments in Proteins. Transworld Research Network, Trivandrum, Vol. 1, pp. 13–26.

    Google Scholar 

  • Collins T, Meuwis MA, Stals I, Claeyssens M, Feller G, Gerday C (2002b) A novel family 8 xylanase: functional and physico-chemical characterization. J Biol Chem 277:35133–35139.

    Article  CAS  PubMed  Google Scholar 

  • Collins T, Meuwis MA, Gerday C, Feller G (2003) Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. J Mol Biol 328:419–428.

    Article  CAS  PubMed  Google Scholar 

  • Collins T, D’Amico S, Marx J, Feller G, Gerday C (2007) Cold-Adapted Enzymes. In: Gerday C, Glansdorff N (eds.), Physiology and biochemistry of extremophiles. ASM Press, Washington, D.C., pp. 165–179.

    Google Scholar 

  • Crawford DL, Powers DA (1992) Evolutionary adaptation to different thermal environments via transcriptional regulation. Mol Biol Evol 9:806–813.

    CAS  PubMed  Google Scholar 

  • D’Amico S, Gerday C, Feller G (2001) Structural determinants of cold adaptation and stability in a large protein. J Biol Chem 276:25791–25796.

    Article  PubMed  Google Scholar 

  • D’Amico S, Marx JC, Gerday C, Feller G (2003) Activity-stability relationships in extremophilic enzymes. J Biol Chem 278:7891–7896.

    Article  PubMed  Google Scholar 

  • D’Amico S, Collins T, Marx JC, Feller G, Gerday C (2006a) Psychrophilic microorganisms: challenges for life. EMBO Rep 7:385–389.

    Article  PubMed  Google Scholar 

  • D’Amico S, Sohier JS, Feller G (2006b) Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase. J Mol Biol 358:1296–1304.

    Article  PubMed  Google Scholar 

  • Davail S, Feller G, Narinx E, Gerday C (1994) Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41. J Biol Chem 269:17448–17453.

    CAS  PubMed  Google Scholar 

  • de Backer M, McSweeney S, Rasmussen HB, Riise BW, Lindley P, Hough E (2002) The 1.9 A crystal structure of heat-labile shrimp alkaline phosphatase. J Mol Biol 318:1265–1274.

    Article  PubMed  Google Scholar 

  • De Vos D, Xu Y, Hulpiau P, Vergauwen B, Van Beeumen JJ (2007) Structural investigation of cold activity and regulation of aspartate carbamoyltransferase from the extreme psychrophilic bacterium Moritella profunda. J Mol Biol 365:379–395.

    Article  PubMed  Google Scholar 

  • Demchenko AP, Ruskyn OI, Saburova EA (1989) Kinetics of the lactate dehydrogenase reaction in high-viscosity media. Biochim Biophys Acta. 998:196–203.

    CAS  PubMed  Google Scholar 

  • Devos N, Ingouff M, Loppes R, Matagne RF (1998) Rubisco adaptation to low temperatures: A comparative study in psychrophilic and mesophilic unicellular algae. J Phycol 34:655–660.

    Article  CAS  Google Scholar 

  • Feller G, Lonhienne T, Deroanne C, Libioulle C, Van Beeumen J, Gerday C (1992) Purification, characterization, and nucleotide sequence of the thermolabile t-amylase from the antarctic psychrotroph Alteromonas haloplanctis A23. J Biol Chem 267:5217–5221.

    CAS  PubMed  Google Scholar 

  • Feller G, Arpigny JL, Narinx E, Gerday C (1997) Molecular adaptations of enzymes from psychrophilic organisms. Comp Biochem Physiol 118:495–499.

    Article  Google Scholar 

  • Feller G (2007) Life at low temperatures: is disorder the driving force? Extremophiles 11:211–216.

    Article  CAS  PubMed  Google Scholar 

  • Fields PA (2001) Review: Protein function at thermal extremes: balancing stability and flexibility. Comp Biochem Physiol A Mol Integr Physiol 129:417–431.

    Article  CAS  PubMed  Google Scholar 

  • Fischer CJ, Schauerte JA, Wisser KC, Gafni A, Steel DG (2000) Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence. Biochemistry. 39:1455–1461.

    Article  CAS  PubMed  Google Scholar 

  • Garcia-Viloca M, Gao J, Karplus M, Truhlar DG (2004) How enzymes work: analysis by modern rate theory and computer simulations. Science 303:186–195.

    Article  CAS  PubMed  Google Scholar 

  • Georlette D, Damien B, Blaise V, Depiereux E, Uversky VN, Gerday C, Feller G (2003) Structural and functional adaptations to extreme temperatures in psychrophilic, mesophilic, and thermophilic DNA ligases. J Biol Chem 278:37015–37023.

    Article  CAS  PubMed  Google Scholar 

  • Georlette D, Blaise V, Collins T, D’Amico S, Gratia E, Hoyoux A, Marx JC, Sonan G, Feller G, Gerday C (2004) Some like it cold: biocatalysis at low temperatures. FEMS Microbiol Rev 28:25–42.

    Article  CAS  PubMed  Google Scholar 

  • Gershenson A, Schauerte JA, Giver L, Arnold FH (2000) Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases. Biochemistry 39:4658–4665.

    Article  CAS  PubMed  Google Scholar 

  • Helland R, Leiros I, Berglund GI, Willassen NP, Smalas AO (1998) The crystal structure of anionic salmon trypsin in complex with bovine pancreatic trypsin inhibitor. Eur J Biochem 256:317–324.

    Article  CAS  PubMed  Google Scholar 

  • Helland R, Larsen AN, Smalas AO, Willassen NP (2006) The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species. FEBS J 273:61–71.

    Article  CAS  PubMed  Google Scholar 

  • Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C (2001) Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis. Appl Environ Microbiol 67:1529–1535.

    Article  CAS  PubMed  Google Scholar 

  • Huston AL, Krieger-Brockett BB, Deming JW (2000) Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice. Environ Microbiol 2:383–388.

    Article  CAS  PubMed  Google Scholar 

  • Karlsen S, Hough E, Olsen RL (1998) Structure and proposed amino-acid sequence of a pepsin from atlantic cod (Gadus morhua). Acta Crystallogr D Biol Crystallogr 54:32–46.

    Article  CAS  PubMed  Google Scholar 

  • Kim SY, Hwang KY, Kim SH, Sung HC, Han YS, Cho Y (1999) Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum. J Biol Chem 274:11761–11767.

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Tsai CJ, Nussinov R (2001) Thermodynamic differences among homologous thermophilic and mesophilic proteins. Biochemistry 40:14152–14165.

    Article  CAS  PubMed  Google Scholar 

  • Leiros I, Moe E, Lanes O, Smalas AO, Willassen NP (2003) The structure of uracil-DNA glycosylase from Atlantic cod (Gadus morhua) reveals cold-adaptation features. Acta Crystallogr D Biol Crystallogr 59:1357–1365.

    Article  PubMed  Google Scholar 

  • Lin JJ, Somero G (1995) Thermal adaptation of cytoplasmic malate dehydrogenases of eastern Pacific barracuda (Sphyraena spp): the role of differential isoenzyme expression. J Exp Biol 198:551–560.

    CAS  PubMed  Google Scholar 

  • Lonhienne T, Gerday C, Feller G (2000) Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim Biophys Acta 1543:1–10.

    CAS  PubMed  Google Scholar 

  • Lonhienne T, Zoidakis J, Vorgias CE, Feller G, Gerday C, Bouriotis V (2001) Modular structure, local flexibility and cold-activity of a novel chitobiase from a psychrophilic Antarctic bacterium. J Mol Biol 310:291–297.

    Article  CAS  PubMed  Google Scholar 

  • Makhatadze GI, Privalov PL (1994) Hydration effects in protein unfolding. Biophys Chem 51:291–309.

    Article  CAS  PubMed  Google Scholar 

  • Makhatadze GI, Privalov PL (1995) Energetics of protein structure. Adv Protein Chem 47:307–425.

    Article  CAS  PubMed  Google Scholar 

  • Mastro AM, Keith AD (1984) Diffusion in the aqueous compartment. J Cell Biol 99:180–187.

    Article  CAS  Google Scholar 

  • Miyazaki K, Wintrode PL, Grayling RA, Rubingh DN, Arnold FH (2000) Directed evolution study of temperature adaptation in a psychrophilic enzyme. J Mol Biol. 297:1015–1026.

    Article  CAS  PubMed  Google Scholar 

  • Narinx E, Baise E, Gerday C (1997) Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein Eng 10:1271–1279.

    Article  CAS  PubMed  Google Scholar 

  • Okubo Y, Yokoigawa K, Esaki N, Soda K, Kawai H (1999) Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus. Biochem Biophys Res Commun 256:333–340.

    Article  CAS  PubMed  Google Scholar 

  • Olufsen M, Smalas AO, Moe E, Brandsdal BO (2005) Increased Flexibility as a Strategy for Cold Adaptation: A comparative molecular dynamics study of cold- and warm-active uracil DNA glycosylase. J Biol Chem 280:18042–18048.

    Article  CAS  PubMed  Google Scholar 

  • Papaleo E, Riccardi L, Villa C, Fantucci P, De Gioia L (2006) Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family. Biochim Biophys Acta 1764:1397–1406.

    CAS  PubMed  Google Scholar 

  • Riise EK, Lorentzen MS, Helland R, Smalas AO, Leiros H-KS, Willassen NP (2007) The first structure of a cold-active catalase from Vibrio salmonicida at 1.96 A reveals structural aspects of cold adaptation. Acta Crystallogr D Biol Crystallogr 63:135–148.

    Article  PubMed  Google Scholar 

  • Russell RJ, Gerike U, Danson MJ, Hough DW, Taylor GL (1998) Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. Structure 6:351–361.

    Article  CAS  PubMed  Google Scholar 

  • Siddiqui KS, Bokhari SA, Afzal AJ, Singh S (2004) A novel thermodynamic relationship based on Kramers Theory for studying enzyme kinetics under high viscosity. IUBMB Life 56:403–407.

    Article  CAS  PubMed  Google Scholar 

  • Siddiqui KS, Feller G, D’Amico S, Gerday C, Giaquinto L, Cavicchioli R (2005) The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase. J Bacteriol 187:6197–6205.

    Article  CAS  PubMed  Google Scholar 

  • Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433.

    Article  CAS  PubMed  Google Scholar 

  • Skalova T, Dohnalek J, Spiwok V, Lipovova P, Vondrackova E, Petrokova H, Duskova J, Strnad H, Kralova B, Hasek J (2005) Cold-active beta-galactosidase from Arthrobacter sp. C2–2 forms compact 660 kDa hexamers: crystal structure at 1.9A resolution. J Mol Biol 353:282–294.

    Article  CAS  PubMed  Google Scholar 

  • Smalas AO, Leiros HK, Os V, Willassen NP (2000) Cold adapted enzymes. Biotechnol Annu Rev 6:1–57.

    Article  CAS  PubMed  Google Scholar 

  • Somero GN (1995) Proteins and temperature. Annu Rev Physiol 57:43–68.

    Article  CAS  PubMed  Google Scholar 

  • Spiwok V, Lipovova P, Skalova T, Duskova J, Dohnalek J, Hasek J, Russell NJ, Kralova B (2007) Cold-active enzymes studied by comparative molecular dynamics simulation. J Mol Model 13:485–497.

    Article  CAS  PubMed  Google Scholar 

  • Suzuki Y, Takano K, Kanaya S (2005) Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli. FEBS J 272:632–642.

    Article  CAS  PubMed  Google Scholar 

  • Svingor A, Kardos J, Hajdu I, Nemeth A, Zavodszky P (2001) A better enzyme to cope with cold. Comparative flexibility studies on psychrotrophic, mesophilic, and thermophilic IPMDHs. J Biol Chem 276:28121–28125.

    Article  CAS  PubMed  Google Scholar 

  • Tehei M, Franzetti B, Madern D, Ginzburg M, Ginzburg BZ, Giudici-Orticoni MT, Bruschi M, Zaccai G (2004) Adaptation to extreme environments: macromolecular dynamics in bacteria compared in vivo by neutron scattering. EMBO Rep 5:66–70.

    Article  CAS  PubMed  Google Scholar 

  • Toyota E, Ng KK, Kuninaga S, Sekizaki H, Itoh K, Tanizawa K, James MN (2002) Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin. J Mol Biol 324:391–397.

    Article  CAS  PubMed  Google Scholar 

  • Tsuruta H, Mikami B, Aizono Y (2005) Crystal structure of cold-active protein-tyrosine phosphatase from a psychrophile, Shewanella sp. J Biochem (Tokyo) 137:69–77.

    CAS  Google Scholar 

  • Uppenberg J, Hansen MT, Patkar S, Jones TA (1994) The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure. 2:293–308.

    Article  CAS  PubMed  Google Scholar 

  • Van Petegem F, Collins T, Meuwis MA, Gerday C, Feller G, Van Beeumen J (2003) The structure of a cold-adapted family 8 xylanase at 1.3 A resolution. Structural adaptations to cold and investigation of the active site. J Biol Chem 278:7531–7539.

    Article  PubMed  Google Scholar 

  • Violot S, Aghajari N, Czjzek M, Feller G, Sonan GK, Gouet P, Gerday C, Haser R, Receveur-Brechot V (2005) Structure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering. J Mol Biol 348:1211–1224.

    Article  CAS  PubMed  Google Scholar 

  • Wang E, Koutsioulis D, Leiros HK, Andersen OA, Bouriotis V, Hough E, Heikinheimo P (2007) Crystal Structure of Alkaline Phosphatase from the Antarctic Bacterium TAB5. J Mol Biol 366:1318–1331.

    Article  CAS  PubMed  Google Scholar 

  • Wintrode PL, Miyazaki K, Arnold FH (2001) Patterns of adaptation in a laboratory evolved thermophilic enzyme. Biochim Biophys Acta. 1549:1–8.

    CAS  PubMed  Google Scholar 

  • Xu Y, Feller G, Gerday C, Glansdorff N (2003a) Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi. J Bacteriol 185:2161–2168.

    Article  CAS  PubMed  Google Scholar 

  • Xu Y, Feller G, Gerday C, Glansdorff N (2003b) Moritella cold-active dihydrofolate reductase: are there natural limits to optimization of catalytic efficiency at low temperature? J Bacteriol 185:5519–5526.

    Article  CAS  PubMed  Google Scholar 

  • Xu Y, Nogi Y, Kato C, Liang Z, Ruger HJ, De Kegel D, Glansdorff N (2003c) Moritella profunda sp. nov. and Moritella abyssi sp. nov., two psychropiezophilic organisms isolated from deep Atlantic sediments. Int J Syst Evol Microbiol 53:533–538.

    Article  CAS  PubMed  Google Scholar 

  • Zavodszky P, Kardos J, Svingor, Petsko GA (1998) Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc Natl Acad Sci USA 95:7406–7411.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Biochemistry, University of Liege, Institute of Chemistry, B6, B-4000, Liege, Belgium

    Tony Collins, Frédéric Roulling, Florence Piette, Jean-Claude Marx, Georges Feller & Salvino D'Amico

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  1. Institute of Microbiology, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria

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  2. Laboratory of Biochemistry, University of Liège, Institute of Chemistry B6, 4000, Liège, Belgium

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Collins, T. et al. (2008). Fundamentals of Cold-Adapted Enzymes. In: Margesin, R., Schinner, F., Marx, JC., Gerday, C. (eds) Psychrophiles: from Biodiversity to Biotechnology. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-74335-4_13

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