Abstract
Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (kcat/Km) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bernfeld P (1955) Amylases α and β. Methods Enzymol 1:149–158
Brown SH, Costantino HR, Kelly RM (1990) Characterization of amylolytic activities associated with the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl Environ Microbiol 56:1985–1991
Costantino HR, Brown SH, Kelly RM (1990) Purification and characterization of an α-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105–115°C. J Bacteriol 172:3654–3660
Dong G, Vieille C, Savchenko A, Zeikus JG (1997a) Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 63:3569–3576
Dong G, Vieille C, Zeikus JG (1997b) Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 63:3577–3584
Erra-Pujada M, Debeire P, Duchiron F, O’Donohue MJ (1999) The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain. J Bacteriol 181:3284–3287
Fujimoto Z, Takase K, Doui N, Momma M, Matsumoto T, Mizuno H (1998) Crystal structure of a catalytic-site mutant α-amylase from Bacillus subtilis complexed with maltopentaose. J Mol Biol 277:393–407
Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316:695–696
Imamura H, Fushinobu S, Jeon BS, Wakagi T, Matsuzawa H (2001) Identification of the catalytic residue of Thermococcus litoralis 4-α-glucanotransferase through mechanism-based labeling. Biochemistry 40:12400–12406
Imamura H, Fushinobu S, Yamamoto M, Kumasaka T, Jeon BS, 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 (1998) Sequence of archaeal Methanococcus jannaschii α-amylase contains features of families 13 and 57 of glycosyl hydrolases: a trace of their common ancestor? Folia Microbiol 43:123–128
Laderman KA, Asaka K, Uemori T, Mukai H, Taguchi Y, Kato I, Anfinsen CB (1993a) α-Amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli. J Biol Chem 268:24402–24407
Laderman KA, Davis BR, Krutzsch HC, Lewis MS, Griko YV, Privalov PL, Anfinsen CB (1993b) The purification and characterization of an extremely thermostable α-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. J Biol Chem 268:24394–24401
MacGregor EA, 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
McCarter JD, Withers SG (1996) Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae α-glucosidase using 5-fluoro glycosyl fluorides. J Biol Chem 271:6889–6894
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Sogaard M, Kadziola A, Haser R, Svensson B (1993) 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. J Biol Chem 268:22480–22484
Vieille C, Zeikus JG (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43
Acknowledgments
This research was supported by a grant from the National Research Initiative Competitive Grants Program, United States Department of Agriculture, under agreement 99-35504-7811.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kang, S., Vieille, C. & Zeikus, J.G. Identification of Pyrococcus furiosus amylopullulanase catalytic residues. Appl Microbiol Biotechnol 66, 408–413 (2005). https://doi.org/10.1007/s00253-004-1690-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-004-1690-7