title: Molecular cloning and biochemical characterization of the first archaeal maltogenic amylase from the hyperthermophilic archaeon Thermoplasma volcanium GSS1.
abstract: Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes belonging to glycoside hydrolase family 13, have been studied extensively, but their physiological roles in microbes and evolutionary relationships with other amylolytic enzymes remain unclear. Here, we report the biochemical properties of a thermostable archaeal MAase from Thermoplasma volcanium GSS1 (TpMA) for the first time. The primary structure and catalytic properties of TpMA were similar to those of MAases, such as possession of an extra domain at its N-terminal and preference for CD over starch. TpMA showed high thermostability and optimal activity at 75 degrees C and 80 degrees C for beta-CD and soluble starch, respectively. The recombinant TpMA exists as a high oligomer in a solution and the oligomeric TpMA was dissociated into dimer and monomer mixture by a high concentration of NaCl. The substrate preference and thermostability of TpMA were significantly dependent on the oligomeric state of the enzyme. However, TpMA exhibited distinguishable characteristics from those of bacterial MAases. The transglycosylation pattern of TpMA was opposite to that of bacterial MAases. TpMA formed more alpha-1,4-glycosidic linked transfer product than alpha-1,6-linked products. Like as alpha-amylases, notably, TpMA has a longer subsite structure than those of other CD-degrading enzymes. Our findings in this study suggest that TpMA, the archaeal MAase, shares characteristics of both bacterial MAases and alpha-amylases, and locates in the middle of the evolutionary process between alpha-amylases and bacterial MAases.
authors.affiliation: Center for Agricultural Biomaterials, and Department of Food Science and Biotechnology, Seoul National University, Seoul 151-921, Korea.
authors.forename: Jung-Woo
authors.initials: JW
authors.lastname: Kim
authors.forename: Yung-Hee
authors.initials: YH
authors.lastname: Kim
authors.forename: Hee-Seob
authors.initials: HS
authors.lastname: Lee
authors.forename: Sung-Jae
authors.initials: SJ
authors.lastname: Yang
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
authors.forename: Myoung-Hee
authors.initials: MH
authors.lastname: Lee
authors.forename: Jung-Wan
authors.initials: JW
authors.lastname: Kim
authors.forename: Nam-Seok
authors.initials: NS
authors.lastname: Seo
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001105:Archaea / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D006031:Glycosylation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D000681:Amylases