title: Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04.
abstract: The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13% sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (Pyrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-β-cyclodextrin (G1-β-CD) and β-cyclodextrin (β-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (Km) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-β-cyclodextrin (G2-β-CD) was 0.3 mM. The kcat values were 381.0 min(-1) for G3 and 1,545.0 min(-1) for G2-β-CD. The enzyme was inhibited competitively by the reaction product G2, and the Ki constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin, 446-701, South Korea.
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Jung
authors.forename: Dong-Ho
authors.initials: DH
authors.lastname: Seo
authors.forename: James F
authors.initials: JF
authors.lastname: Holden
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D008970:Molecular Weight; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D008320:Maltose; D000516:alpha-Amylases