Construction and characterization of chimeric enzymes of kojibiose phosphorylase and trehalose phosphorylase from Thermoanaerobacter brockii.
Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.
Yamamoto
Takuo
T
Glycoscience Institute, Research Center, Hayashibara Biochemical Laboratories, Inc., 675-1 Fujisaki, Okayama 702-8006, Japan. takyamamoto@hayashibara.co.jp
Yamashita
Hiroshi
H
Mukai
Kazuhisa
K
Watanabe
Hikaru
H
Kubota
Michio
M
Chaen
Hiroto
H
Fukuda
Shigeharu
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D004187:Disaccharides / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D006005:Phosphorylases / Q000138:chemical synthesis*; D017434:Protein Structure, Tertiary; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology*; D014312:Trisaccharides / Q000302:isolation & purification
D004187:Disaccharides; D014312:Trisaccharides; C014637:laminaribiose; D005964:Glucosyltransferases; D006005:Phosphorylases; C506185:kojibiose phosphorylase, Thermoanaerobacter brockii; C023181:alpha,alpha-trehalose phosphorylase