Biologia 65/3: 408—415, 2010 Section Cellular and Molecular Biology DOI: 10.2478/s11756-010-0030-x Tyrosine 39 of GH13 α-amylase from Thermococcus hydrothermalis contributes to its thermostability Andrej Godány 1,2 , Katarína Majzlová 1 , Viera Horváthová 2 , Barbora Vidová 1 & Štefan Janeček 1,2 * 1 Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava, Slovakia; e-mail: Stefan.Janecek@savba.sk 2 Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, SK-91701 Trnava, Slovakia 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. Key words: α-amylase; Thermococcus hydrothermalis; glycoside hydrolase family 13; site-directed mutagenesis; protein thermostability. Abbreviations: GH, glycoside hydrolase. Introduction α-Amylases (EC 3.2.1.1) are enzymes capable in general to catalyze the hydrolysis of the α-1,4-glycosidic bonds in starch and related poly- and oligo-saccharides. They are produced by various organisms originating from all the three domains of life – Bacteria, Archaea and Eu- carya (Janecek 2005; MacGregor 2005; Sivaramakrish- nan et al. 2006; Seo et al. 2008). From the primary structure point of view, there are two families of α- amylases within the sequence-based classification of gly- coside hydrolases (GHs) at the CAZy server (Cantarel et al. 2009): the family GH13 and the family GH57. The two types of α-amylases do not only differ from each other by their amino acid sequences but also by their catalytic machineries and tertiary structures (e.g., Matsuura et al. 1984; Kadziola et al. 1994; Uitdehaag et al. 1999; MacGregor et al. 2001; Imamura et al. 2003; Linden et al. 2003; Robert et al. 2003; Zona et al. 2004; Ballschmiter et al. 2006; Dickmanns et al. 2006; Sivaku- mar et al. 2006; Tan et al. 2008). On the other hand the α-amylases from both families GH13 and GH57 employ the same retaining reaction mechanism of the glycosidic bond cleavage (McCarter & Withers 1994). With regard to α-amylases from the family GH13, known currently as the α-amylase family (MacGregor et al. 2001), at the beginning of 1990s, several groups were recognized, such as: (i) fungi and yeasts; (ii) plants; (iii) animals and actinomycetes; and (iv) the rest of bacteria that were scattered in the evolutionary tree (Janecek 1994). No α-amylase from an archaeon was known at that time and the plant α-amylases were clus- tered with liquefying and intracellular counterparts of bacterial origin (Janecek 1994). The situation dramati- cally changed when the first archaeal GH13 α-amylases were sequenced, e.g. those from Thermococcus sp. Rt3 (Jones et al. 1999) and Thermococcus hydrothermalis (Leveque et al. 2000a), and were surprisingly found at * Corresponding author c 2010 Institute of Molecular Biology, Slovak Academy of Sciences