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.
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Abbreviations
- GH:
-
glycoside hydrolase
References
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.
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.
Bernfeld P. 1955. Amylases, α and β. Methods Enzymol. 1: 149–158.
Bertoldo C. & Antranikian G. 2002. Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr. Opin. Chem. Biol. 6: 151–160.
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.
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.
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.
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.
Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.
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.
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.
Janecek S. 1994. Sequence similarities and evolutionary relationships of microbial, plant and animal α-amylases. Eur. J. Biochem. 224: 519–524.
Janecek S. 2002. How many conserved sequence regions are there in the α-amylase family? Biologia 57(Suppl. 11): 29–41.
Janecek S. 2005. Amylolytic families of glycoside hydrolases: focus on the family GH-57. Biologia 60(Suppl. 16): 177–184.
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.
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.
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.
Kadziola A., Abe J., Svensson B. & Haser R. 1994. Crystal and molecular structure of barley α-amylase. J. Mol. Biol. 239:104–121.
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.
Leveque E., Janecek S., Belarbi A. & Haye B. 2000b. Thermophilic archaeal amylolytic enzymes. Enzyme Microb. Technol. 26: 2–13.
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.
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.
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.
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.
MacGregor E.A. 2005. An overview of clan GH-H and distantly related families. Biologia 60(Suppl. 16): 5–12.
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.
Matsuura Y., Kusunoki M., Harada W. & Kakudo M. 1984. Structure and possible catalytic residues of Taka-amylase A. J. Biochem. 95: 697–702.
McCarter J.D. & Withers S.G. 1994. Mechanisms of enzymatic glycoside hydrolysis. Curr. Opin. Struct. Biol. 4: 885–892.
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.
Page R.D. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12: 357–358.
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.
Saitou N. & Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425.
Savchenko A., Vieille C., Kang S. & Zeikus J.G. 2002. Pyrococcus furiosus α-amylase is stabilized by calcium and zinc. Biochemistry 41: 6193–6201.
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.
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.
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.
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.
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.
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.
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.
Vieille C. & Zeikus G.J. 2001. Hyperthermophilic enzymes: sources, uses and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65: 1–43.
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.
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Godány, A., Majzlová, K., Horváthová, V. et al. Tyrosine 39 of GH13 α-amylase from Thermococcus hydrothermalis contributes to its thermostability. Biologia 65, 408–415 (2010). https://doi.org/10.2478/s11756-010-0030-x
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DOI: https://doi.org/10.2478/s11756-010-0030-x