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Báo cáo khoa học: Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis

Fifty-nine amino acid sequences belonging to family 57 (GH-57) of the glycoside hydrolases were collected using the CAZy server, Pfamdatabase andBLASTtools. 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-a-glucanotransferase from Thermococcus lito-ralis, these motifs correspond to 13_HQP (region I), 76_GQLEIV (region II), 120_WLTERV (region III), 212_HDDGEKFGVW (region IV), and 350_AQCNDA YWH (region V). . | Eur. J. Biochem. 271 2863-2872 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04144.x Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis Richard Zona1 I Florent Chang-Pi-Hin2 I Michael J. O Donohue2 and Stefan Janecek1 Institute of Molecular Biology member of the Centre of Excellence for Molecular Medicine Slovak Academy of Sciences Bratislava Slovakia 2Institut National de la Recherche Agronomique UMR FARE Reims France 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-a-glucanotransferase from Thermococcus lito-ralis these motifs correspond to 13_HQP region I 76_GqLEIV region II 120_WLTERV region Ill 212_hDdGEKFGVW region IV and 350_AQCNDA YWH 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 amylopullu-lanase. Therefore these data support the prediction and strongly suggest that the bifunctionality of the amylopullu-lanase 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 .