Figure 3
The complete nucleotide sequences and deduced amino acid sequences of DzAmy1 (GenBank accession no. KU212896). The numbers correspond to the mature DzAmy1 sequence. The catalytic triad (Asp179, Glu204, and Asp281) is marked with bold and italic letters. The start of the mature protein and stop (TAA) codon are represented with italic letters and an asterisk, respectively. The putative signal peptide is indicated by underlined letters.
Figure 4
The amino acid sequence alignment of DzAmy1 and other plant α-amylases. Residues labelled with M indicate the start (Gln25) of the mature protein at the N-terminus of barley Amy2 (KADZIOLA et al., 1994 KADZIOLA, A.; ABE, J.; SVENSSON, B.; HASER, R. Crystal and molecular structure of barley α-amylase. Journal of Molecular Biology, Amsterdam, v.239, p.104-121, 1994. ). Residues conserved in starch hydrolase are in marked with S. Residues labelled in close proximity to three calcium ions in barley α-amylase (KADZIOLA et al., 1994 KADZIOLA, A.; ABE, J.; SVENSSON, B.; HASER, R. Crystal and molecular structure of barley α-amylase. Journal of Molecular Biology, Amsterdam, v.239, p.104-121, 1994. ) are marked with C. Residues labelled with N are those that have hydrogen bond interactions with the acarbose inhibitor and are near the active site in barley α-amylase (KADZIOLA et al., 1998 KADZIOLA, A.; SOGAARD, M.; SVENSSON, B.; HASER, R. Molecular structure of a barley α-amylase inhibitor complex: implications for starch binding and catalysis. Journal of Molecular Biology, Amsterdam, v.278, p. 205–217, 1998. ). Residues labelled with G and Y are in the starch granule binding site (SOGAARD et al., 1993 SOGAARD, M.; KADZIOLA, A.; HASER, R.; SVENSSON, B. Site-directed mutagenesis of histidine 93, aspartic acid 180, glutamic acid 205, histidine 290, and aspartic acid 291 at the active site and tryptophan 279 at the raw starch binding site in barley α-amylase 1. Journal of Biological Chemistry, Baltimore, v.268, p.22480–22484, 1993. ) and sugar binding site (ROBERT et al., 2003 ROBERT, X.; HASER, R.; GOTTSCHALK, T.E.; RATAJCZAK, F.; DRIGUEZ, H.; SVENSSON, B.; AGHAJARI N. 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, London, v.11, p.973–984, 2003. ) in barley α-amylase. The catalytic residues (Asp, Glu and Asp) are highlighted with bold letters and grey shading.
Figure 5
Evolutionary tree analysis and tertiary structure of DzAmy1 by homology modelling. The plant α-amylase phylogenetic tree analysis. The tree was constructed by using the MEGA5 program (TAMURA et al., 2011 TAMURA, K.; PETERSON, D.; PETERSON, N.; STECHER, G.; NEI, M.; KUMAR, S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, Oxford, v.28, p.2731–2739, 2011. ) with the neighbour-joining method (SAITOU et al., 1987 SAITOU, N.; NEI, M. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, Oxford, v.4, p.406-425, 1987. ). The bootstrap values were derived from 1,000 bootstrap replicates, and the values are shown as percentages. The plant α-amylases used in this study are as follows: Durio zibethinus Murr. cv. Mon Thong, Durian DzAmy1 (KU212896), Durian DzAmy3 (KP164993); Malus domestica, Apple Amy10 (AAX33231), Apple Amy8 (AAF63239), Apple Amy9 (AAX33234), Apple Amy2 (AAX33232); Actinidia chinensis, Kiwifruit (AAX33233); Musa acuminata, Banana (AAN01149); Manihot esculenta, Cassava Amy2 (AAY85174); Hordeum vulgare, Barley Amy2 (AAA32925), Barley Amy1 (AAA32927), Barley Amy2-2 (AAA98790), Barley Amyaa24 (CAA72143); Oryza sativa, Rice AmyA (AAA33885), Rice Amy3B (AAA33897), Rice AmyB (AAA33886); Phaseolus vulgaris, French bean Amy1 (BAA33879); Solanum tuberosum, Potato Amy21 (AAA91883), Potato Amy23 (AAA91884), Triticum aestivum, Wheat Amy3 (AAA34259), Zea mays, Maize Amy1 (AAA50161); Vigna mungo, Black gram Amy1 (CAA37217); Aspergillus oryzae (AAA32708) .
Figure 6
Three-dimensional structure modelling of DzAmy1. (a) Three-dimensional structure modelling of DzAmy1 that displays as a ribbon structure of a-helices and ß-strands composed of three domain (domain A (red), B (blue) and C (yellow)). The calcium ion represents as a green sphere. (b) Structural characterization of DzAmy1 using X-ray crystal structure of 2.8 Å resolution barley α-amylase AMY2 (1BG9) as a template (KADZIOLA et al., 1998 KADZIOLA, A.; SOGAARD, M.; SVENSSON, B.; HASER, R. Molecular structure of a barley α-amylase inhibitor complex: implications for starch binding and catalysis. Journal of Molecular Biology, Amsterdam, v.278, p. 205–217, 1998. ). The superimposed structures of DzAmy1 (blue) and 1BG9 (yellow) are shown as ribbon structures. The acarbose bound at the catalytic cleft and starch-granule binding sites (disaccharide fragment) are shown as grey sticks. Important amino acids are highlighted as sticks (DzAmy1, blue; 1BG9, yellow). Calcium ions are represented as purple spheres.