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Volume 65, Issue 3 (Jun 2010)

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Tyrosine 39 of GH13 α-amylase from Thermococcus hydrothermalis contributes to its thermostability

Andrej Godány
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, SK-91701, Trnava, Slovakia
  • Email:
/ Katarína Majzlová
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Email:
/ Viera Horváthová
  • Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, SK-91701, Trnava, Slovakia
  • Email:
/ Barbora Vidová
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Email:
/ Štefan Janeček
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, SK-91701, Trnava, Slovakia
  • Email:
Published Online: 2010-04-24 | DOI: https://doi.org/10.2478/s11756-010-0030-x

Abstract

The presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.

Keywords: α-amylase; Thermococcus hydrothermalis; glycoside hydrolase family 13; site-directed mutagenesis; protein thermostability

  • [1] Bairoch A., Bougueleret L., Altairac S., Amendolia V., ... & Zhang J. 2009. The Universal protein resource (UniProt) 2009. Nucleic Acids Res. 37(Database issue): D169–D174.

  • [2] Ballschmiter M., Fütterer O. & Liebl W. 2006. Identification and characterization of a novel intracellular alkaline α-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl. Environ. Microbiol. 72: 2206–2211. http://dx.doi.org/10.1128/AEM.72.3.2206-2211.2006 [Crossref]

  • [3] Bernfeld P. 1955. Amylases, α and β. Methods Enzymol. 1: 149–158. http://dx.doi.org/10.1016/0076-6879(55)01021-5 [Crossref]

  • [4] Bertoldo C. & Antranikian G. 2002. Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr. Opin. Chem. Biol. 6: 151–160. http://dx.doi.org/10.1016/S1367-5931(02)00311-3 [Crossref]

  • [5] Cantarel B.L., Coutinho P.M., Rancurel C., Bernard T., Lombard V. & Henrissat B. 2009. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37(Database Issue): D233–D238. http://dx.doi.org/10.1093/nar/gkn663 [Crossref]

  • [6] Da Lage J.L., Feller G. & Janecek S. 2004. Horizontal gene transfer from Eukarya to Bacteria and domain shuffling: the α-amylase model. Cell. Mol. Life Sci. 61: 97–109. http://dx.doi.org/10.1007/s00018-003-3334-y [Crossref]

  • [7] Declerck N., Machius M., Joyet P., Wiegand G., Huber R. & Gaillardin C. 2002. Engineering the thermostability of Bacillus licheniformis α-amylase. Biologia 57(Suppl. 11): 203–211.

  • [8] Dickmanns A., Ballschmiter M., Liebl W. & Ficner R. 2006. Structure of the novel α-amylase AmyC from Thermotoga maritima. Acta Crystallogr. D Biol. Crystallogr. 62: 262–270. http://dx.doi.org/10.1107/S0907444905041363 [Crossref]

  • [9] Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. http://dx.doi.org/10.2307/2408678 [Crossref]

  • [10] Horvathova V., Godany A., Sturdik E. & Janecek S. 2006. α-Amylase from Thermococcus hydrothermalis: re-cloning aimed at the improved expression and hydrolysis of corn starch. Enzyme Microb. Technol. 39: 1300–1305. http://dx.doi.org/10.1016/j.enzmictec.2006.03.016 [Crossref]

  • [11] Imamura H., Fushinobu S., Yamamoto M., Kumasaka T., Jeon B.S., Wakagi T. & Matsuzawa H. 2003. Crystal structures of 4-α-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor. J. Biol. Chem. 278: 19378–19386. http://dx.doi.org/10.1074/jbc.M213134200 [Crossref]

  • [12] Janecek S. 1994. Sequence similarities and evolutionary relationships of microbial, plant and animal α-amylases. Eur. J. Biochem. 224: 519–524. http://dx.doi.org/10.1111/j.1432-1033.1994.00519.x [Crossref]

  • [13] Janecek S. 2002. How many conserved sequence regions are there in the α-amylase family? Biologia 57(Suppl. 11): 29–41.

  • [14] Janecek S. 2005. Amylolytic families of glycoside hydrolases: focus on the family GH-57. Biologia 60(Suppl. 16): 177–184.

  • [15] Janecek S. 2008. Sequence fingerprints in the evolution of the α-amylase family, pp. 45–63. In: Park K.H. (Ed.) Carbohydrate-Active Enzymes: Structure, Function and Applications. Woodhead Publishing, Ltd., Cambridge.

  • [16] Janecek S., Leveque E., Belarbi A. & Haye B. 1999. Close evolutionary relatedness of α-amylases from Archaea and plants. J. Mol. Evol. 48: 421–426. http://dx.doi.org/10.1007/PL00006486 [Crossref]

  • [17] Jones R.A., Jermiin L.S., Easteal S., Patel B.K. & Beacham I.R. 1999. Amylase and 16S rRNA genes from a hyperthermophilic archaebacterium. J. Appl. Microbiol. 86: 93–107. http://dx.doi.org/10.1046/j.1365-2672.1999.00642.x [Crossref]

  • [18] Kadziola A., Abe J., Svensson B. & Haser R. 1994. Crystal and molecular structure of barley α-amylase. J. Mol. Biol. 239:104–121. http://dx.doi.org/10.1006/jmbi.1994.1354 [Crossref]

  • [19] Leveque E., Haye B. & Belarbi A. 2000a. Cloning and expression of an α-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterisation of the recombinant enzyme. FEMS Microbiol. Lett. 186: 67–71. http://dx.doi.org/10.1016/S0378-1097(00)00117-8 [Crossref]

  • [20] Leveque E., Janecek S., Belarbi A. & Haye B. 2000b. Thermophilic archaeal amylolytic enzymes. Enzyme Microb. Technol. 26: 2–13. http://dx.doi.org/10.1016/S0141-0229(99)00142-8 [Crossref]

  • [21] Liebl W., Stemplinger I. & Ruile P. 1997. Properties and gene structure of the Thermotoga maritima α-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium. J. Bacteriol. 179: 941–948.

  • [22] Lim J.K., Lee H.S., Kim Y.J., Bae S.S., Jeon J.H., Kang S.G. & Lee J.H. 2007. Critical factors to high thermostability of an α-amylase from hyperthermophilic archaeon Thermococcus onnurineus NA1. J. Microbiol. Biotechnol. 17: 1242–1248.

  • [23] Linden A., Mayans O., Meyer-Klaucke W., Antranikian G. & Wilmanns M. 2003. Differential regulation of a hyperthermophilic α-amylase with a novel (Ca, Zn) two-metal center by zinc. J. Biol. Chem. 278: 9875–9884. http://dx.doi.org/10.1074/jbc.M211339200 [Crossref]

  • [24] Lowry O.H., Rosebrough N.I., Farr A.L. & Randall R.I. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193: 265–275.

  • [25] MacGregor E.A. 2005. An overview of clan GH-H and distantly related families. Biologia 60(Suppl. 16): 5–12.

  • [26] MacGregor E.A., Janecek S. & Svensson B. 2001. Relationship of sequence and structure to specificity in the α-amylase family of enzymes. Biochim. Biophys. Acta 1546: 1–20.

  • [27] Matsuura Y., Kusunoki M., Harada W. & Kakudo M. 1984. Structure and possible catalytic residues of Taka-amylase A. J. Biochem. 95: 697–702.

  • [28] McCarter J.D. & Withers S.G. 1994. Mechanisms of enzymatic glycoside hydrolysis. Curr. Opin. Struct. Biol. 4: 885–892. http://dx.doi.org/10.1016/0959-440X(94)90271-2 [Crossref]

  • [29] Nelson K.E., Clayton R.A., Gill S.R., Gwinn M.L., ... & Fraser C.M. 1999. Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima. Nature 399: 323–329. http://dx.doi.org/10.1038/20601 [Crossref]

  • [30] Page R.D. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12: 357–358.

  • [31] Robert X., Haser R., Gottschalk T.E, Ratajczak F., Driguez H., Svensson B. & Aghajari N. 2003. The structure of barley α-amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs. Structure 11: 973–984. http://dx.doi.org/10.1016/S0969-2126(03)00151-5 [Crossref]

  • [32] Saitou N. & Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425. [Web of Science]

  • [33] Savchenko A., Vieille C., Kang S. & Zeikus J.G. 2002. Pyrococcus furiosus α-amylase is stabilized by calcium and zinc. Biochemistry 41: 6193–6201. http://dx.doi.org/10.1021/bi012106s [Crossref]

  • [34] Seo E.S., Christiansen C., Abou Hachem M., Nielsen M.M., Fukuda K., Bozonnet S., Blennow A., Aghajari N., Haser R. & Svensson B. 2008. An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans. Biologia 63: 967–979. http://dx.doi.org/10.2478/s11756-008-0164-2 [Crossref] [Web of Science]

  • [35] Sivakumar N., Li N., Tang J.W., Patel B.K. & Swaminathan K. 2006. Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly-extreme condition. FEBS Lett. 580: 2646–2652. http://dx.doi.org/10.1016/j.febslet.2006.04.017 [Crossref]

  • [36] Sivaramakrishnan S., Gangadharan D., Nampoothiri K.M., Soccol C.R. & Pandey A. 2006. α-Amylases from microbial sources — an overview on recent developments. Food Technol. Biotechnol. 44: 173–184.

  • [37] Tan T.C., Mijts B.N., Swaminathan K., Patel B.K. & Divne C. 2008. Crystal structure of the polyextremophilic α-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch. J. Mol. Biol. 378: 852–870. http://dx.doi.org/10.1016/j.jmb.2008.02.041 [Crossref] [Web of Science]

  • [38] Thompson J.D., Higgins D.G. & Gibson T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680. http://dx.doi.org/10.1093/nar/22.22.4673 [Crossref]

  • [39] Uitdehaag J.C., Mosi R., Kalk K.H., van der Veen B.A., Dijkhuizen L., Withers S.G. & Dijkstra B.W. 1999. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family. Nat. Struct. Biol. 6: 432–436. http://dx.doi.org/10.1038/8235 [Crossref]

  • [40] van der Kaaij R.M., Janecek S., van der Maarel M.J. & Dijkhuizen L. 2007. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes. Microbiology 153: 4003–4015. http://dx.doi.org/10.1099/mic.0.2007/008607-0 [Crossref] [Web of Science]

  • [41] Vieille C. & Zeikus G.J. 2001. Hyperthermophilic enzymes: sources, uses and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65: 1–43. http://dx.doi.org/10.1128/MMBR.65.1.1-43.2001 [Crossref]

  • [42] Zona R., Chang-Pi-Hin F., O’Donohue M.J. & Janecek S. 2004. Bioinformatics of the family 57 glycoside hydrolases and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis. Eur. J. Biochem. 271: 2863–2872. http://dx.doi.org/10.1111/j.1432-1033.2004.04144.x [Crossref]

About the article

Published Online: 2010-04-24

Published in Print: 2010-06-01



Citation Information: Biologia, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-010-0030-x. Export Citation

© 2010 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. (CC BY-NC-ND 3.0)

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