Fifty-nine amino acid sequences belonging to family 57 (GH-57) of the glycoside hydrolases were collected using the CAZy server, Pfam database and BLAST tools. Owing to the sequence heterogeneity of the GH-57 members, sequence alignments were performed using mainly manual methods. Likewise, five conserved regions were identified, which are postulated to be GH-57 consensus motifs. In the 659 amino acid-long 4-α-glucanotransferase from Thermococcus litoralis, these motifs correspond to 13_HQP (region I), 76_GQLEIV (region II), 120_WLTERV (region III), 212_HDDGEKFGVW (region IV), and 350_AQCNDAYWH (region V). The third and fourth conserved regions contain the catalytic nucleophile and the proton donor, respectively. Based on our sequence alignment, residues Glu291 and Asp394 were proposed as the nucleophile and proton donor, respectively, in a GH-57 amylopullulanase from Thermococcus hydrothermalis. To validate this prediction, site-directed mutagenesis was performed. The results of this work reveal that both residues are critical for the pullulanolytic and amylolytic activities of the amylopullulanase. Therefore, these data support the prediction and strongly suggest that the bifunctionality of the amylopullulanase is determined by a single catalytic centre. Despite this positive validation, our alignment also reveals that certain GH-57 members do not possess the Glu and Asp corresponding to the predicted GH-57 catalytic residues. However, the sequences concerned by this anomaly encode putative proteins for which no biochemical or enzymatic data are yet available. Finally, the evolutionary trees generated for GH-57 reveal that the entire family can be divided into several subfamilies that may reflect the different enzyme specificities.